KR20010015817A - Modified Antibodies with Enhanced Ability to elicit an anti-idiotype response - Google Patents

Abstract

The present invention relates to modified immunoglobulin molecules, wherein one or more mutant regions forming interchain disulfide bonds are replaced with amino acid residues having no SH (sulfydryl) group so that no interchain disulfide bonds are formed. These immunoglobulin molecules have the ability to induce enhanced anti-genic responses. The present invention also provides methods of preventing and treating cancer and infectious diseases using the modified immunoglobulins of the present invention.

Description

Modified Antibodies with Enhanced Ability to elicit an anti-idiotype response

2. Background of the Invention

2.1. Immunoglobulin structure

The basic unit of immunoglobulin structure is four polypeptide complexes, in which two identical low molecular weight or "light" (L) chains and two identical high molecular weight or "heavy" (H) chains are used for non-covalent association and disulfide bonds. Are connected by. Each L and H of the antibody has a variable portion at its amino acid terminus and always has a portion at its C terminus (see FIG. 1). The variable portion is unique for each antibody and includes an antibody antigen binding site. Each variable domain consists of four related conserved structures and three parts of complementarity determining parts or hypervariable sequences called CDRs (FIG. 2). In most cases it is the CDRs that form the antigen binding site and provide antigen specificity. The part is always very conservative compared to the variable domain, with some diversity due to the haplotype difference.

Based on their amino acid sequences, L chains are classified as either kappa or lambda. The H chain always consists of multiple domains (CH1, CH2, CH3 ... CHx), the number of which depends on the specific antibody type. The CH1 moiety is separated from CH2 with a hinge moiety interposed between them that provides flexibility to the antibody. The variable portion of each L chain is arranged in line with the variable portion of each H chain, and the always portion of each L chain is arranged in line with the first always portion of each H chain. The CH2-CHx domain of the always part of the H chain makes an "Fc part", which binds to Fc receptors expressed by complementary binding, lymphocytes, granule cells, unicellular lineage cells, killer cells, plaque cells and other immune effector cells. It is in charge of the function of the effective substance.

As can be seen in FIG. 3, each L and H chain of the IgG molecule creates a variable partial domain and always a partial domain. Each domain consists of two protein layers of about 100 amino acids in length, sandwiched by a single disulfide bond (Roitt et al., Immunology, 3rd Edition, London; Mosby, 1993, p 4.4). Each domain of two protein layers contains two "anti-paralle" contiguous strands to form a beta-sheet shape (e.g., Stryer, 1975, Biochemistry, WH Freeman and Co., p. 950). Each domain has a similar tertiary structure based on immunoglobulin folds.

2.2. Immunotherapy and Anti-idiotype Antibodies

According to modern medicine, immunotherapy or vaccination has resulted in the elimination of diseases such as polio, tetanus, tuberculosis, chicken pox, measles and hepatitis. Vaccination reactions were used to investigate the ability of the immune system to prevent infectious diseases.

The study was carried out using immunotherapy for the treatment of cancer. The field of tumor immunology was initiated by Prehn and Main, which showed that the antigens were tumor specific for sarcomas induced by methylchloranthrene (MCA), which was not detected in normal tissues of rats by transplantation tests. (Prehn et al., 1957, J. Natl. Cancer Inst. 18: 79-778). This was confirmed by further experiments demonstrating that tumor specific resistance to MCA-induced tumors was induced in spontaneous hosts, i.e., mice from which the tumor originated (Klein et al., 1990, cancer Res. 20: 151-1572).

There are several reasons why it is desirable to use immunotherapy to treat cancer patients. First, if cancer patients are immunosuppressed in anesthesia and subsequent chemotherapy surgery, immunosuppression can be exacerbated, and then appropriate immunotherapy during the preoperative period, such immunosuppression is inhibited or reversed. Can be. This reduces complications and accelerates wound healing. Second, tumor size is reduced after surgery, and immunotherapy is most effective in this situation. Third, because tumor cells enter the circulatory system in surgery, effective immunotherapy at this time can remove these cells.

There are two types of immunotherapy, "active immunotherapy" and "passive immunotherapy". In "active immunotherapy", the antigen is administered in the form of a vaccine [vaccination] to induce a protective immune response. "Passive immunotherapy" is the administration of an antibody to a patient without inducing an immune response at the same time. When a specific antibody taken in one animal is injected with an immunogen into an appropriate second animal, the injected antibody will induce an immune response. Antibody therapy is passive because the patient is not the source of the antibody. However, the term passive is misleading because the patient makes an anti-idiotype second antibody, which causes an immune response that has cross reactivity with the native antigen. Immunotherapy, which makes a second antibody in a patient, is therapeutically more effective than passive immunotherapy because, after the initial infusion of the antibody, cells bearing a specific antigen and the patient's native immune system are constantly fighting.

In an anti-idiotypic response, an antibody initially made or introduced into an organism has a unique novel epitope that the organism is not resistant to, and induces second antibody production (“Ab2”). Some of these will be against the idiotype (antigen binding site) of the first antibody (“Ab1”), which is an antibody initially introduced or produced externally. Such a second antibody or Ab2 has an idiotype capable of inducing a third antibody (“Ab3”), some of which may recognize the antigen binding site of Ab2. This is known as the so-called "network" theory. Some of the second antibodies have a binding moiety that is similar to the native antigen, and thus can recreate an "internal image" of the native antigen. Thus, a third or Ab3 antibody that recognizes the antigen binding site of the Ab2 antibody may also recognize the native antigen (FIG. 4).

Thus, anti-idiotype antibodies have binding sites similar to the shape and charge of the antigen and can induce a response equal to or greater than that of the cancer antigen itself. Administration of exogenous antibodies that can induce potent anti-idiotype responses, induces a constant immune response and acts as an effective vaccine.

To date, anti-idiotype vaccines consist of murine antibodies because the anti-idiotype response occurs as part of a typical human anti-mouse antibody (HAMA) response. A strong anti-idiotype step was observed when structurally disrupting Ab1 (Madiyalakan et al., 1995, Hybridoma 14: 199-203) to impart more heterogeneity to the antibody. The subject is exogenously administered directly with an anti-idiotype antibody raised against the idiotype of the anti-tumor antibody (U.S. Patent No. 4,918,14). After administration, anti-antibodies are made in the body of the individual, which not only recognizes these anti-idiotype antibodies, but also the native tumor epitopes, in relation to complementary activation and other immune system responses to foreign bodies. Attack tumor cells expressing epitopes.

However, although anti-idiotype vaccines are a desirable target and some have been identified, the ability to carry antibodies that can repeatedly hear such anti-idiotype responses is currently unavailable (foon et al., 1995, J. Clin, Invest. 9: 334-342; Madiyalakan et al., 1995, Hybridoma 14: 199-203). One of the reasons why such anti-idiotype reactions cannot be made is because exogenous Ab1 is very similar to the very similar structure of all antibodies, so the anti-idiotype response to itself is very limited. Thus, there is a need in the art for methods that can reliably anti-idiotype responses to specific antibodies.

3. Summary of the Invention

The present invention replaces one or more variable moiety cysteine residues of an antibody that can form one or more interchain disulfide bonds with amino acid residues that do not have an SH group, so that the antibody that does not form a particular disulfide bond has disulfide bonds in a unique variable moiety. It is based on the inventors' finding that they can induce more potent anti-idiotype responses than antibodies.

Accordingly, the present invention provides a vaccine composition comprising a modified immunoglobulin molecule or antibody (and functionally active fragments, derivatives and analogs thereof) and such immunoglobulin molecules, wherein the variable region of the immunoglobulin is one The dissociation of disulfide bonds between the chains or in the chains reduces the binding due to shape. Specifically, the present invention provides a modified immunoglobulin consisting of the same variable moiety except having one or more amino acid substituted moieties in the variable moiety for the second immunoglobulin molecule, wherein the second immunoglobulin molecule is immune to the antigen. Non-specific binding, such as specific binding of an immunoglobulin to an antigen such as determined by any method known in the art for determining antibody-antigen binding, such as Excluded, and not cross-reactive with other antigens, wherein one or more amino acid substituents are substituted with one or more amino acid residues that do not have an SH group at one or more positions corresponding to one or more cysteine residues that make disulfide bonds in the second immunoglobulin molecule . In suitable embodiments, the second immunoglobulin molecule is capable of immunospecifically binding to a cancer antigen; In another suitable embodiment, the second immunoglobulin molecule may immunospecifically bind to an antigen of an infectious disease medium or a cell receptor of the infectious disease medium.

The present invention also provides a method of inducing an anti-idiotype response in a subject by administering to the subject a modified immunoglobulin of the invention. In certain embodiments, a modified immunoglobulin molecule of the invention is used to treat and prevent cancer, wherein the immunoglobulin molecule is immune capable of immunospecific binding to a cancer antigen expressed in relation to a particular cancer patient. From the globulin molecule, for example, a modified amino acid residue which does not contain an SH group in accordance with the present invention is replaced with a cysteine residue of one or more variable moieties that make disulfide bonds in the chain. Furthermore, in other embodiments, the modified immunoglobulin molecules of the invention are used to treat or prevent an infectious disease, for example immunospecific to cell receptors or antigens in an infectious disease medium that is responsible for the infectious disease. An immunoglobulin molecule derived from an immunoglobulin molecule capable of binding can be administered.

The invention also provides a method of producing a modified immunoglobulin molecule of the invention and a vaccine composition comprising the modified immunoglobulin molecule of the invention.

Related applications

This application claims the advantages of preliminary application 60 / 065,716 filed November 14, 1997 and pre-application 60 / 081,403 filed April 10, 1998.

1. Field of Invention

The present invention relates to modified immunoglobulins and vaccine compositions thereof, wherein in modified immunoglobulins, one or more variable partial cysteine residues forming disulfide bonds in the chain are replaced with amino acid residues that do not contain an SH group, thereby not disulfide bond formation. . The invention also relates to the use of the vaccine compositions of the invention for the treatment and prevention of certain diseases, in particular cancer and infectious diseases.

Figure 1 shows the structure of the L and H chains of an immunoglobulin molecule, each chain consisting of a variable portion at the amino terminal portion (H ₂ N-) and an always portion at the carboxy terminal portion (-COOH).

Figure 2 shows the structure of IgG, with four basic portions (FR1, FR2, FR3, FR4) and three complementarity determining portions (CDR1, CDR2, CDR3) in the variable portions (V _L, V _H ) of each L and H chain. ) Is present. Always partial domains are represented by the L chain always part as C _L and the three domains of the H chain always part as CH _1, CH _2, CH ₃ . FAb is represented as part of an antibody fragment, which includes the variable partial domains of the L and H chains and the C _L and CH ₁ domains. Fc always refers to a partial fragment comprising the CH ₂ and CH ₃ domains.

FIG. 3 shows a diagram of the antibody structure shown in FIG. 2, in which each domain (V _L, V _H, C _L, CH _1, CH _2, CH ₃ is indicated in the loop structure, respectively) is disulfide bond (black line) ), Which is structurally defined and maintains a tertiary structure (Roitt et al., Immunology, Second Edition, London: Gower Medical Publishig, 1989, p 5.3).

4 shows the generation of a home image with an anti-idiotype antibody (Ab2) and an anti-anti-idiotype antibody (Ab3) from an idiotype antibody (Ab1) generated for a ligand in an anti-idiotype step. A diagram illustrating the process.

5 shows modification of the variable portion of an immunoglobulin by replacing cysteine residues in the variable portion with alanine residues to break up disulfide bonds in the variable portion chain. CH1, CH2, CH3 are always part. V _H becomes an H chain variable moiety, and V _L represents an L chain variable moiety.

(A) in FIGS. 6A-C (A) show the structure of the expression vector pMRRO10.1 comprising the human kappa L chain always partial sequence. (B) shows the structure of the expression vector pGamma1 comprising a sequence encoding the human IgG1 always part (CH1, CH2, CH3) H chain and the hinge part sequence. (C) shows the structure of the expression vector pNEPuDGV comprising a sequence encoding the kappa always domain of the L chain and the always domain and hinge portion of the H chain. For these three vectors, see Bebbington et al., 1991, Methods in Enzymology 2: 136-145.

(A) in FIGS. 7A and B show the amino acid sequence and the corresponding nucleotide sequence comprising the leader sequence of the consensus L chain variable portion ConVL1. (B) shows the amino acid sequence for the consensus H chain variable portion CONVL1 and the corresponding nucleotide sequence comprising the leader sequence.

In Figures 8A-B, (A) is the amino acid and corresponding nucleotide sequence of 2CAVLCOL1, the L chain variable subsequence of the antibody derived from mAb31.1, wherein the alanine residues are substituted for cysteine residues at positions 23 and 88 It is marked with a box. (B) is the amino acid and corresponding nucleotide sequence of 2CAVHCOL1, the H chain variable subsequence of the antibody derived from mAb31.1, wherein the alanine residues are substituted for cysteine residues at positions 23 and 88 and are boxed It is.

In Figures 9A-D (A) shows the oligonucleotide sequence of the oligonucleotides used to assemble 2CAVHCOL1, which is an H chain variable partial gene specific for human colon cancer antigens. (B) shows the oligonucleotide sequence of the oligonucleotides used to assemble 2CAVHCOL1, which is an L chain variable partial gene specific for human colon cancer antigens. (C) shows the oligonucleotide sequence for the oligonucleotide used to assemble the L chain consensus portion referred to as ConVL1. (D) shows the oligonucleotide sequence for the oligonucleotide used to assemble the H chain consensus portion referred to as ConVL1.

10 shows the overall steps of assembling an engineered gene encoding a modified modified antibody specific for a human determinant cancer antigen.

FIG. 11 shows dot blots showing the results of testing binding competition between cysteine derived from mAB31.1 unaltered with alanine and the same antibody biotin-labeled to antigen secretions derived from LS-174 T-cells. The concentration of unlabeled antibody is shown as nM unlabeled antibody. The "blk" line is free of antigen.

(A) in Figures 12A-D are reference antibodies that bind to biotin-labeled anti-colon carcinoma cell antibodies and unmodified colon carcinoma cell antibodies to LS-174T in the presence of anti-serum of mice vaccinated with carrier only and Competitive binding test results of CDR1, CDR2, CDR3, CDR4, CDR5, CDR6 peptides having CDR sequences comprising a bradykinin receptor binding site expressed in LS-174T cells at baseline binding ratios are shown. (B) shows competition of biotin-labeled anti-colon carcinoma cell antibodies on LS-174T cells with unmodified 2CAVHCOL1 and 2CAVLCOL1 colon cancer cell antibodies in the presence of anti-serum of mice vaccinated with carrier alone. The results of the binding test are shown. (C) is a diagram showing binding of a biotin-labeled (represented by "b") antibody (inverted Y-shape) to an antigen (dark triangle). (D) shows that biotin-labeled (represented by "b") antibody (inverted Y-shaped) to antigen (dark triangle) is prevented from binding by anti-idiotype antibody (arrow).

13 shows the nucleotide sequence of an L chain variable moiety having a CDR comprising a binding sequence for HNFG1.

The present invention provides modified immunoglobulins (particularly antibodies, functionally active fragments, derivatives, analogs thereof) that can induce potent immune responses, in particular potent anti-idiotype responses, compared to unmodified immunoglobulins. In particular, the modified immunoglobulins of the invention, when unmodified, are modified to immunospecifically bind to the antigen and to reduce shape-induced binding to one variable portion of the immunoglobulin molecule, preferably the variability of the immunoglobulin At least one of the cysteines involved in forming intrachain disulfide bonds in the moiety is substituted with amino acid residues that do not have an SH group, resulting in no disulfide bonds, thereby reducing shape-restriction in at least one part of the variable moiety of the immunoglobulin. (FIG. 5). In a suitable embodiment of the invention, the modified immunoglobulin molecule is derived from an immunoglobulin molecule capable of immunospecifically binding to a cancer antigen, and in another suitable embodiment, the modified immunoglobulin molecule is an infectious disease medium or infectious It is derived from immunoglobulins capable of immunospecific binding to cellular receptors on disease portals.

The present invention also provides a vaccine composition comprising the modified immunoglobulin molecule of the invention. The invention also provides a method of generating an anti-idiotype response by administering a modified immunoglobulin molecule of the invention to a subject.

In certain embodiments, the present invention provides a method of treating or preventing cancer by administering a modified immunoglobulin molecule of the invention, wherein in an unmodified state it is able to specifically bind to a cancer antigen that is expressed in association with a particular cancer. have. Administration of the modified immunoglobulin induces an anti-idiotype response in the subject, thereby producing an antibody specific for the cancer antigen in the subject. In another specific embodiment, the modified immunoglobulin may bind to an antigen of a cellular receptor to an infectious disease medium or infectious disease medium in an unmodified state. Such immunoglobulins are used to treat or prevent infectious diseases caused by an infectious disease medium.

In the following description, for the sake of clarity, the present invention is described in several subparagraphs, but is not intended to limit the present invention.

5.1. Modified antibody

Modified immunoglobulins, particularly antibodies, of the invention are immunoglobulins that specifically bind antigens, at least in unmodified conditions, and are modified in a way that enhances their ability to induce anti-idiotype responses. Such immunoglobulins can be modified to reduce constraints due to the shape in the variable portion of the immunoglobulin, for example to remove or reduce disulfide bonds or chemically modify or to disulfide bonds within or within chains. Use the method. Specifically, the present invention provides a first immunoglobulin molecule consisting of the same variable moiety except for one or more amino acid substitutions in the variable part of the second immunoglobulin, wherein the second immunoglobulin molecule is immunospecific to the antigen. Capable, amino acid substitutions are those that are substituted with one or more amino acid residues having no SH group at positions corresponding to one or more cysteine residues that form disulfide bonds in the second immunoglobulin molecule. The invention also provides a nucleic acid comprising a nucleotide sequence encoding a modified immunoglobulin of the invention.

Methods known in the art can be used to identify cysteine residues that can form disulfide bonds in the variable portion of a particular antibody. For example, cysteine residues that form intrachain disulfide bonds are known to be highly conserved between antibody types and between species. Thus, cysteine residues that participate in forming disulfide bonds can be identified by comparing sequences with other antibody moieties known as residues that disulfide bonds.

Table 1 provides the location of cysteine residues that can form disulfide bonds in a large number of antibody molecules (Kabat et al, 1991, sequences of Proteins of Immunological Interest, 5th Ed., US Department of Health and Human Services, Bethesda). , Maryland)

Table 1

Numbers indicating positions in () are residues estimated by comparison with known sequences for which no sequence was identified at this position.

Notably, in all antibody molecules listed in Table 1, the cysteine residues that form intrachain disulfide bonds are at positions 22 and 88 of the L chain variable domain and at positions 22 and 92 of the H chain variable domain. The number indicating the position refers to a residue that corresponds to a residue in the consensus sequence described by Kabat (1991, Sequences of Proteins of Immunological Interest, 5th Ed, US Department of Health and Human Services, Bethesda, Maryland) or FIG. 7A. And residues such as those shown in the H and L chain variable region sequences described in B ("corresponding" means those of the H and L chain variable regions as shown in FIG. 7A or B or specific antibody sequences of the consensus sequence). Means arranged by side by side).

Thus, in one embodiment of the invention, the modified immunoglobulin molecule is substituted with an amino acid residue whose residue at position 23 or 88 of the L chain has no SH group or the residue at position 22 or 92 of the H chain has SH group. Refers to an antibody substituted with an amino acid residue.

In the modified immunoglobulins of the present invention, amino acids replacing cysteine residues that form disulfide bonds are any amino acid residues that do not have an SH group, for example, alanine, arginine, asparagine, aspartate (or aspartic acid). , Glutamine, glutamate (or glutamic acid), glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine. In certain embodiments, cysteine residues are substituted with glycine, serine, threonine, tyrosine, asparagine, glutamine residues, most preferably with alanine residues.

In addition, cysteines that form disulfide bonds may be substituted by, but not limited to, non-traditional amino acids or chemical amino acid analogues that do not include SH groups, such as made by routine protein synthesis methods. Non-traditional amino acids include D-isomers of conventional amino acids, α-amino acid isobutyl acid, 4-aminobutyl acid, Abu, r-Abu, 2-aminobutyl acid, γ-Abu, ε-Ahx, amino hexanoic acid, Aib, 2-amino isobutyl acid, 3-amino propionic acid, ornithine, norruic acid, norvaline, hydroxyproline, sarcosine, cyturulin, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine , but are not limited to, β-alanine, fluoro-amino acids, β-methyl amino acids, Cα-methyl amino acids, designer amino acids such as Nα-methyl amino acids, common amino acid derivatives, and the like. In addition, the amino acids may be of type D or L. In another embodiment, residues that form disulfide bonds may be removed.

In certain embodiments, residue substitutions that form disulfide bonds can be in the H chain variable moiety or the L chain variable moiety or in both the H and L chain variable moiety. In other specific embodiments, one of the residues that form a particular disulfide bond is substituted (or deleted) or all of the residues that form a particular disulfide bond are substituted (deleted).

In another embodiment, the invention relates to a second immunoglobulin molecule that forms a disulfide bond in the variable moiety, wherein the invention is substituted with an amino acid that does not include an SH group and further comprises one or more other amino acid substituents (eg, disulfide bonds). Residues to be formed are not substituted with residues that do not contain an SH group).

In particular, the present invention provides a first immunoglobulin molecule having a variable moiety consisting of the same as the second immunoglobulin molecule except for having at least one amino acid substituent in the variable moiety for the second immunoglobulin molecule, wherein the second An immunoglobulin molecule may immunospecifically bind to an antigen, wherein at least one of the one or more amino acid substituents does not have an SH group at a position corresponding to one or more cysteine residues that form disulfide bonds in the second immunoglobulin molecule Replaced with.

In a preferred embodiment, amino acid substituents in which the cysteine residues forming disulfide bonds are not substituted with residues having no SH group are not stable changes. Stable changes are defined as amino acid changes that increase the stability of the antibody molecule. Such amino acid changes are amino acids that are not customary at specific positions for a particular antibody molecule (such as those provided by Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., US Department of Health and Human Services, Bethesda Maryland, Is defined as the consensus sequence for a number of antibody molecules, which is replaced by substitution with common residues at specific positions (PCT Publication WO96 / 02574, data February 1, 1996 by Steipe et al.).

Such other amino acid substitutions are any amino acid substitutions that do not modify the modified immunoglobulin to induce anti-anti-idiotype antibody formation, which can be determined by the method described in 5.5. For example, such other amino acid substituents include functionally equivalent amino acid residue substituents. For example, one or more amino acid residues are substituted with another amino acid residue having a similar polarity that is functionally equivalent. Amino acid substitutions in the sequence are selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine, and the like. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine and the like. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

Modified antibodies of the invention can be derived from antibodies that are capable of immunospecifically binding to any antigen. In certain embodiments, the modified antibody is derived from an antibody capable of immunospecifically binding to a cancer antigen, more suitably a tumor antigen. In certain embodiments, the modified antibody is a polymorphic epithelial mucosal antigen, a human colon carcinoma-associated protein antigen, a human colon carcinoma-associated carbohydrate antigen, an antibody capable of binding to human fat or breast cancer, ovarian cancer, uterine cancer, bladder cancer Is derived from an antibody capable of binding to an antigen of lung cancer, skin cancer, colon cancer, pancreatic cancer, gastrointestinal cancer, B lymphocyte cancer or T lymphocyte cancer or an antigen of any other cancer expressing a specific antigen described in paragraph 5.2.1. . In a suitable embodiment, the modified antibody comprises MAb31.1 (Accession No. 12314 of the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2201), Mab 33.28 (Accession No. 12315), Mab HMFG-1 ( see PCT Publication WO90 / 05142 and PCT Publication WO92 / 04380).

In another specific embodiment, the antibody is capable of immunospecifically binding to an antigen of an infectious disease medium or a cellular receptor of an infectious disease medium. In a suitable embodiment, the antigen for the medium of an infectious disease is any other antigen, such as a bacterial antigen, a viral antigen or a parasite antigen or an infectious disease medium described in 5.2.2.

The immunoglobulin molecules of the invention can be any kind, class or subclass of immunoglobulins. In suitable embodiments, the immunoglobulin molecule is of a type selected from IgG molecule IgE, suitably IgG, IgE, IgM, IgD, IgA, most suitably an IgG molecule. Or the immunoglobulin molecule becomes an unmodified-domain of T cell receptor or B cell receptor, cell expressing adsorption molecule such as co-receptor CD4, CD8, CD19 or MHC molecule.

Modified immunoglobulins can be derived from any naturally occurring antibody, suitably monoclonal antibodies, or from synthetic or engineered antibodies. In one aspect of the invention the modified immunoglobulin molecule can be derived from an antibody incorporated in one or all of the CDRs in which the binding site for a member of the binding pair or binding site for a portion of the antigen is in the variable region ( This can be seen in co-pending US patent application "Immunoglobulin Molecules Having A Synthetic Variable Region And Modified Specificity", and Burch, Filed November 13, 1998 (attorney docket no. 6750-016).

In particular, a synthetic antibody is an antibody capable of immunospecifically binding to a first member of a binding pair, wherein at least one of the CDRs of the antibody in the binding pair comprises a binding site of the first member of the binding pair, wherein the binding site Is derived from the amino acid sequence of another member of the binding pair. In one aspect of the invention, the amino acid sequence of the binding site is not in nature in the CDRs. In addition, at least one of the CDRs comprises a portion of the antigen, in particular an epitope.

The amino acid sequence of the binding site can be confirmed by any method known in the art. For example, in some cases, it has already been identified that the sequence of a member of a binding pair is directly related to the binding of other members of the binding pair. In such cases, such sequences can be used to construct CDRs of synthetic antibodies that specifically recognize other members of a binding pair. If the amino acid sequence for the binding site is not known to one member of the binding pair for another member of the binding pair, any method known in the art can be used, for example, by molecular modeling or empirical methods, such as a member The sequence can be determined by examining a portion of (e.g., a peptide) or by making a mutation in a member, determining whether the mutation interferes with binding, and the like.

The binding pair may be any two molecules that can interact with each other, such as proteins, nucleic acids, carbohydrates, lipids, and the like. In suitable embodiments, the modified immunoglobulin comprises a binding sequence for a cancer antigen, an infectious disease antigen, a cell receptor for a pathogen, a cell receptor for a ligand or a receptor participating in a receptor-ligand binding pair.

In certain embodiments, the binding pair is a homogeneous interaction (eg, interaction between two identical proteins) or heterologous interaction (eg, interaction between two different proteins) in a protein-protein interaction pair.

In certain embodiments, the first member is a member of a ligand-receptor binding pair, suitably a member of a receptor-ligand binding pair, where the ligand binds to the receptor and induces a physiological response such as intracellular signaling. For example, a ligand or receptor may be a hormone, autocoid, growth factor, cytokine, neurotransmitter or any receptor or ligand that is involved in the receptor or signal delivery of a hormone, autocoid, growth factor, cytokine, neurotransmitter. Signal transduction pathways, see, eg, Campbell, 1997, J. Pediat, 131: S42-S44; Hamilton, 1997, J. Leukoc. Biol. 62: 145-155; Soede-Bobok & Touw, 1997, J. Mol. Med. 75: 47-477; 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, 1991, Cell 64: 281-302). In certain embodiments, one member of the binding pair is cholecytokine, galanine, IL-1, IL-2, IL-4, IL-5, IL-6, IL-11, chemokine, leptin, protease, Neuropeptide Y, Neurokinin-1, Neurokinin-2, Neurokinin-3, Bombesin, Castrin, Corticotropin-releasing hormone, Endotherin, Metaltonin, Smatostatin, Vasoactive visceral peptide, Epithelium Ligands such as growth factors, tumor necrosis factor, dopamine, endothelin, or the like, or any receptor of these igands. In one embodiment, one member of the binding pair comprises an opioid receptor, a glucose transporter, a glutamate receptor, an orphanin receptor, an erythropoietin receptor, an insulin receptor, a tyrosine kinase (TK) -receptor, a KIT hepatocellular factor receptor, a nerve Receptors such as growth factor receptors, insulin-like growth factor receptors, granulocyte-colony stimulating factor receptors, somatotropin receptors, glial-induced neuronal factor receptors or gp39 receptors, G-protein receptors or β2-adrenergic receptors or Arbitrary ligands that bind to these receptors, and the like. In another embodiment, one of the members of the binding pair is, but is not limited to, ligand gate ion channels such as calcium channels, sodium channels, potassium channels, and the like. In certain embodiments, the present invention provides a modified immunoglobulin that immunospecifically binds to a receptor, which is an antagonist of a ligand that binds to the receptor, for example, an antagonist of endorphin, enkephalin, or nocisepin. It is not limited to this. In another embodiment, the present invention is a modified antibody that immunospecifically binds to a receptor, which is an antagonist of the receptor, for example, but is not limited to, antagonists of endorphins, enkephalins, noscipine. In suitable embodiments, the modified immunoglobulin does not bind to the fibronectin receptor. In another suitable embodiment the binding sequence is not Arg-Gly-Asp, not a multiple binding sequence, preferably the binding sequence is not Arg-Gly-Asp.

In another specific embodiment, the modified immunoglobulin has a CDR that includes a binding site for a transcription factor. In a suitable aspect, the modified immunoglobulin does not bind to specific DNA sequences, in particular it does not bind to transcriptional binding recognition.

In a suitable embodiment the modified immunoglobulin has at least one CDR comprising the amino acid sequence of a binding site to a cancer antigen or a tumor antigen (see description in 5.2.1), more preferably the antigen is a human colon carcinoma-associated antigen. Or epithelial mucosal antigen. In another embodiment, at least one CDR of the modified immunoglobulin comprises an amino acid sequence for the binding site of a human milk fat receptor. In another embodiment, the modified immunoglobulin comprises the amino acid sequence of the binding site of the tumor antigen of the breast, ovary, uterus, prostate, bladder, lung, skin, pancreas, colon, gastrointestinal tract, B-lymphocytes, T-lymphocytes. Include.

In another suitable embodiment of the present invention, at least one CDR of the modified antibody has a binding site for the antigen of the infectious disease medium (see description in paragraph 5.2.2) or a binding site of the cell receptor of the infectious disease medium, appropriately. Preferably the binding site is not the amino acid sequence of the Plasmodium antigen or has the amino acid sequence of the binding site which is not Asn-Ala-Asn-Pro or Asn-Val-Asp-Pro. In further embodiments, the modified antibody has a CDR comprising a binding site for a bacterial or viral enzyme.

Synthetic antibodies can be made based on the sequences of existing or naturally occurring antibodies (eg, by inserting binding site sequences into the CDRs of such antibodies) or synthesized from known antibody consensus sequences, which are illustrated in FIG. It can be synthesized from L and H antibody consensus sequences at 7A and B or from any other antibody consensus or germline sequences (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5 ^th edition, NIH). Publication No. 91-3242, pp 2147-2172).

Each antibody molecule has six CDR sequences, three in the L chain and three in the H chain, five of which are germline CDRs (eg, derived directly from the animal's genomic sequence without any recombination). And one of the CDRs is a non-germline CDR (eg, different from the germline genome sequence of an animal and is generated by recombination of the germline sequence). Whether a CDR is a germline or non-germline sequence is determined by examining the sequence of the CDR and comparing it with known germline sequences (Kabat et al., (1991, Sequences of Proteins of Immunological Interest, 5 ^th). edition, NIH Publication No. 91-3242, pp 147-2172). Significant changes in known germline sequences indicate that the CDRs are non-germline CDRs. Thus, the CDRs comprising the amino acid sequence of the binding site or antigen are germline CDRs, otherwise non-germline CDRs.

Inserting a binding site or antigen sequence into any CDRs of an antibody and inserting different CDRsd [binding sites of the antibody are known in the art and then modified antibodies generated for its ability to bind to specific members of the binding pair ( Paragraph 5.5) or a modified antibody (Dock 5.5) generated for its ability to induce an immune response against the antigenic site. Thus, one skilled in the art can determine whether the CDRs optionally comprise a binding site or antigen. In certain embodiments, the CDRs of the H or L chain are modified to include the amino acid sequence of the binding site or antigen. In another specific embodiment, the modified antibody comprises a variable domain that binds to the first, second, third CDRs of the variable portion of the H chain or to the first, second, third CDRs of the variable portion of the L chain. Amino acid portion of the site or antigen. In another embodiment of the invention, one or more CDRs comprise the binding site or amino acid sequence of an antigen, or one or more CDRs each comprise different binding sites of the same molecule or different binding sites of another molecule. In particular, two, three, four, five, six CDRs are engineered to have the binding site of the first member of the binding pair. In suitable embodiments, one or more CDRs comprise a binding site for the first member of the binding pair, and the immune cells, such as T cells, B cells, NK cells, K cells, The binding site of the molecule on the surface of the TIL cell. For example, by using a modified antibody having a binding site for a cancer antigen or an infectious disease antigen and a binding site for a molecule on the surface of the immune cell, the immune cell may target a cancer cell or infectious disease medium having a cancer antigen. Do it.

In certain embodiments of the invention, the binding site or antigen amino acid sequence is inserted into the CDR without any change to the amino acid sequence of the CDR itself or replaces the binding site or antigen amino acid sequence with all or all of the CDR. In certain embodiments, the binding site amino acids replace the 1,2,5,8,10,15,20 amino acids of the CDR sequences.

The amino acid sequence of the binding site or antigen in the CDRs is the minimum binding site necessary for binding to the members of the binding pair or for inducing an immune response against the antigen (determined by any known method); Or the amino acid sequence of the binding site or antigen is larger than the minimum binding site or antigen sequence necessary for binding to a member of the binding pair or required to elicit an immune response against the antigen. In certain embodiments, the binding site or antigenic amino acid sequence is at least 4 amino acids in length or at least 6,8,10,15,20 amino acids in length. In another embodiment, the binding site amino acid sequence is 10,15,20, 25 amino acids in length or 5-10,5-15,5-20,10-15,10-20,10-25 amino acids in length .

In addition, the total length of the CDRs (eg, the length of the binding site sequence plus the remaining CDR sequences) should be an appropriate number of amino acids to allow the antibody to bind to the antigen. It can be seen that the CDRs have a specific range of amino acid residues, the size of the identified CDRs (also indicated in Figure 2) are shown in Table 2.

TABLE 2

Many CDR H3 moieties are 5-9 residues in length, while certain CDR H3 moieties are much longer. In particular, many anti-viral antibodies have H chain CDR H3 moieties that are 17-24 residues in length.

Thus, in certain embodiments of the invention, the CDRs comprising the binding site or antigenic portion are in the size range provided in the particular CDRs in Table 2, e.g., the initial CDRs of the L chain, i. Having from 17 amino acid residues, for the second CDR of the L chain, L2, the CDRs have 7 amino acid residues, and for the third CDR of the L chain, L3, the CDRs have 7-11 amino acid residues. Have The first CDR of the H chain, i.e. CDR, has 5 to 7 amino acid residues, and in the case of the second CDR of the H chain, H2, the CDR has 9 to 12 amino acid residues and the third of the H chain. In the case of a CDR, ie H3, the CDRs have from 2 to 25 amino acid residues. In another specific embodiment, the CDRs comprising a binding site have amino acids 5-10,5-15,5-20,11-15,11-20,11-25, 16-25 in length. In other embodiments, the CDRs having a binding site have at least 5, 10, 15, 20 amino acids or 10, 15, 20, 25, 30 amino acids in length.

After constructing the antibody comprising the modified CDRs, the modified antibody is further modified and screened for antibodies with greater affinity or specificity. Antibodies with greater affinity or specificity for the target antigen are made and selected by any method known in the art. For example, a nucleic acid encoding a synthesized modified antibody may be mutated randomly or by a chemical or site-directed mutagenesis or specific mutagenesis at a specific position in a nucleic acid encoding a modified antibody, to a target antigen. Antibodies exposed to mutated nucleic acid molecules with binding affinity are selected. Selection is performed by individually testing the expressed antibody molecules or by screening libraries of mutated sequences such as phase display techniques (US 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.).

In certain embodiments, the invention provides functionally active fragments, derivatives or analogs of the modified immunoglobulin molecules of the invention. Functionally active means that fragments, derivatives, and analogs can recognize the same antigen that the antibody from which they are derived is capable of inducing anti-anti-idiotype antibodies (eg, tertiary antibodies or Ab3 antibodies). (This can be verified by the method described in paragraph 5.5). In particular, in suitable embodiments, eliminating the CDR sequences and structures that are the N-terminus of a particular CDR sequence that specifically recognizes an antigen enhances the antigenicity of the idiotype of an immunoglobulin molecule. To determine if the CDR sequences bind to the antigen, synthetic peptides comprising the CDR sequences are used for antigen binding assays, which use any binding assay known in the art. Thus, in a suitable embodiment, the invention encompasses modified immunoglobulin molecules in which a cysteine residue forming one disulfide bond in the variable moiety domain is replaced with an amino acid residue not comprising an SH group and a portion of the variable moiety is deleted. do.

Other embodiments of the invention include fragments of the modified antibodies of the invention, including, for example, F (ab ') ₂ comprising a variable moiety, the L chain always part and the CH1 domain of the H chain made by pepsin treatment of the antibody molecule. And Fab fragments produced by reducing the disulfide bridges of F (ab ') ₂ , but are not limited thereto. The present invention also provides any minimum fragments, such as the H and L chain dimers or Fvs or single chain antibodies (SCAs) of the modified molecules of the invention or any other molecule with the same specificity as the modified antibodies of the invention ( eg, as described in US Patent 4,946,778; Bird, 1988, Science 242: 423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 87: 5879-5883; and Ward et al., 1989, Nature 334: 544-54).

Techniques for producing "chimeric antibodies" have also been developed (Morrison et al., 1984, Proc. NA, Acad. Sci. 81: 851-855; Neuberger et al., 1984, Nature 312: 604-608, Takeda) et al., 1985, Nature 314: 452-454), conjugating the genes of mouse antibody molecules with appropriate antigen specificity to those of human antibody molecules with appropriate biological activity. Chimeric antibodies refer to, for example, humanized antibodies with molecules having different portions derived from different animal species, such as those having variable portions derived from murine monoclonal antibodies, and those having the always part of human immunoglobulins.

In certain embodiments, the modified immunoglobulins of the invention are those of a humanized antibody, more suitably the structural portion of a human antibody, and the CDRs have a variable domain derived from an antibody of a non-human animal suitably mouse ( International Patent Application No. PCT / GB8500392 by Neuberger et al. And Celltech Limited).

CDR conjugation is another method of humanized antibodies. This is a remaking of murine antibodies, which conveys the overall antigen specificity and binding affinity to the human structure (Winter et al. U. S. Patnet No. 5,225, 539). CDR-conjugated antibodies have been successfully made against a variety of antigens, for example, antibodies against IL-2 receptors (Queen et al., 1989 (Proc. NA C. Acad. Sci. USA 86: 10029); cells Antibody to Surface Receptor-CAMPATH (Riechmann et al. (1988, Nature, 332: 323)); Antibody to Hepatitis B (Cole et al. (1991, Proc. Natl. Acad. Sci. USA 88: 2869)) Antibodies against antigen-respiratory conjugate viruses (Tempest et al. (1991, Bio-Technology 9: 267)), etc. CDRs-conjugated antibodies are made by conjugating CDRs of murine monoclonal antibodies to human antibodies. , Benefiting from further amino acid changes in the structural moiety to maintain affinity since the skeletal residues are essential for maintaining the CDR shape and some framework residues are known to be part of the antigen binding site. However, in order to preserve the framework structure in order not to introduce any antigenic sites, Compared to established germline sequences and computer modeled.

In another embodiment, the present invention provides a fusion protein of the modified immunoglobulin of the invention (or a functionally active fragment thereof), eg, via covalent linkage of a modified immunoglobulin, which is not modified immunoglobulin. Is fused to the N- or C-terminus of the amino acid sequence of another protein (or a portion thereof, at least a portion of 10, 20, 50 amino acids of the protein). Suitably the modified immunoglobulin or fragment thereof is always covalently linked to another protein at the N-terminus of the portion. In suitable embodiments, the present invention provides a modified immunoglobulin wherein IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, γ-interferon, MCH derived peptide, G-CSF, It is a fusion protein covalently linked to a TNF, porin, NK cell antigen or cellular endocytosis receptor.

Modified immunoglobulins of the present invention include modified analogs and derivatives, including, for example, covalently attached to any type of molecule, provided that such covalent binding induces an anti-idiotype response, It should not interfere with the modified immunoglobulin (which is determined by any method described in paragraph 5.5). For example, derivatives and analogs of the modified immunoglobulins of the invention include those that are further modified, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, known And those derived by methods such as protecting and blocking groups, proteolytic cleavage, linkage to cellular ligands, or any portion thereof. Any of a variety of chemical modifications may be made using known techniques, including but not limited to cleavage with specific chemicals, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. . In addition, analogs or derivatives may include one or more non-crossing amino acids, such as those listed in this paragraph.

Methods for making modified immunoglobulins, fragments, analogs and derivatives thereof are described in 5.4.

5.2. Therapeutic Uses

The present invention provides a method of inducing anti-idiotype antibody and anti-anti-idiotype antibody production by administering a therapeutic agent to a subject. Such therapeutic agents include modified immunoglobulins of the invention, functionally active fragments, analogs and derivatives thereof (see paragraph 5.1), nucleic acids encoding modified antibodies of the invention, functionally active fragments thereof, derivatives thereof (See paragraph 5.1).

In general, it is desirable to administer a product with a botanical or species reactivity that is the same species as the individual. Thus, in suitable embodiments, the methods of the present invention utilize modified antibodies derived from human antibodies, and in still other embodiments use modified antibodies derived from chimeric or humanized antibodies.

In particular, a vaccine composition comprising the modified antibody of the invention (paragraph 5.3) is administered to a subject to produce an antibody (eg, an anti-idiotype antibody or Ab2) that specifically recognizes the idiotype of the modified antibody Ab2. Inducing, again, this leads to the production of an anti-anti-idiotype antibody (Ab3) that specifically recognizes the idiotype of Ab2, such that the Ab3 antibody has the same or similar specificity as the modified antibody.

The present invention provides a method of administering a modified antibody of the invention to induce an anti-idiotype response, such as to make Ab2 and Ab3 type antibodies. Alternatively, the present invention provides an Ab3-type antibody in a second individual by administering a modified antibody of the invention to produce an Ab2 antibody in one individual, isolating the Ab2 antibody, and then administering an Ab2 antibody to a second individual. Provide a way to make it.

Accordingly, the present invention provides a method of inducing an anti-idiotype response in a subject, the method comprising a first immunoglobulin molecule (or a functionally active fragment thereof or sufficient thereof) that induces an anti-idiotype response. Derivative) and the first immunoglobulin has the same variable moiety as the second immunoglobulin molecule, except that there is at least one amino acid residue substituent in the variable moiety, and the second immunoglobulin molecule is immune to the antigen. One or more substituents having the ability to bind specifically, wherein one or more substituents are substituted with amino acid residues that do not contain an SH group at one or more positions corresponding to one or more cysteine residues that form disulfide bonds in the second immunoglobulin molecule Has In another embodiment, there is further provided a method of isolating an anti-idiotype antibody that recognizes the idiotype of a second immunoglobulin molecule and administering the anti-idiotype antibody to the second individual.

In certain embodiments, which will be discussed in more detail in the following paragraphs, the modified antibodies of the present invention may be used to include, but are not limited to, infectious agents, diseased or abnormal cells such as bacteria, parasites, fungi, viruses, tumors, and cancers. Do not induce an anti-idiotype response. The modified antibodies of the invention can be used to induce an anti-anti-idiotype response to a specific antigen, thereby treating or preventing any disease that can be treated or prevented.

In another embodiment, the modified antibody is used to treat autoimmune diseases, including but not limited to the treatment of rheumatoid arthritis, lupus, ulcerative colitis, psoriasis or allergies, and the like. In one specific embodiment, the methods and compositions of the invention are used to induce a humoral response in a subject. In another embodiment, the methods and compositions of the invention are used to induce cell-mediated responses in a subject. In suitable embodiments, the methods and compositions of the present invention are used to induce humoral and cell-mediated responses.

Individuals that can utilize the present invention are any mammalian or vertebrate species, for example, cattle, horses, sheep, pigs, poultry (chicken), goats, cats, dogs, helmsters, mice, rats, monkeys, rabbits. This includes, but is not limited to, chimpanzees and humans. In suitable embodiments, the subject is a human. Using the compositions and methods of the present invention, diseases are prevented or certain diseases are treated, wherein an anti-idiotype response to a particular immunoglobulin molecule is effective for treating or preventing the disease.

5.2.1. Cancer Treatment and Prevention

Cancers, including any disease characterized by neoplasia, tumors, metastases or unregulated cell growth, are nucleic acids encoding modified immunoglobulins (or active fragments, derivatives and analogs thereof) or modified immunoglobulins thereof or Can be treated and prevented by administering functionally active fragments, derivatives and analogs,

Such modified immunoglobulins are derived from immunoglobulins that specifically recognize one or a plurality of antigens, which antigens are associated with cancer cells of the cancer to be treated or prevented. Whether a particular therapy is effective in preventing or treating some form of cancer can be determined by any method known in the art, examples of which are provided in Section 5.5, but are not limited to these examples.

For example, cancers associated with the following cancer antigens are treated or prevented by administering the modified antibodies of the invention derived from antibodies recognizing these cancer antigens: KS1 / 4 pan-cancer (species) antigens (Perez and Walker, 1990, J. Immunol. 142: 32-37; bumal, 1988 Hybridoma7 (4): 407-415), ovarian cancer antigen (CA125) (Yu et al., 1991, Cancer Res. 51 (2): 48-475), Prostate 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 p97 (Estin et al., 1989, J. Natl. Cancer Instit. 81 (6): 445-44), Melanoma antigen gp75 (vijayasardahl et al., 1990. J. Exp. Med. 171 (4): 1375-1380), high molecular weight melanoma antigen (HMW-MAA) (Natali et al., 1987, Cancer 59:55 -3; Mittelman et al., 1990, j. Chin.invest 86: 2136-2133). Prostate specific membrane antigen, fetal cancer (species) antigen (CEA) (Foon et al., 1994, Proc. Am. Soc. Clin. Oncol. 13: 294), polymorphic endothelial murine antigen, human milk fat antigen, colon Rectal Cancer-Related Antigens (e.g. CEA, TAG-72 (Yokata et al., 1992, Cancer Res. 52: 3402-3408), C017-1A (Ragnhammar et al., 1993, Int, J. Cancer 53: 751-) 758); GICA 19-9 (Herlyn et al., 1982, J. Clin. Immunol. 2: 135), CTA-1, LEA), bulk kit lymphoma antigen 38.13, CD19 (Ghetie et al., 1994, Blood 83 : 1329-1336), human b-lymphoma antigen-CD20 (Reff et al., 1994, Blood 83: 435-445), CD33 (Sgouros et al., 1993, J. Nucl. Med. 34: 422-430) , Melanoma specific antigens (e.g., strong glycid GD2 (Saleh et al., 1993, J. Immunol. 151,3390-3398), strong glycid GD3 (Shitara et al., 1993, Cancer Immunol. Immunother, 36) : 373-380), strong glycid GM2 (Livingston et al., 1994, J. Clin. Oncol. 12: 1036-1044), strong glycid GM3 (Hoon et al., 1993, Cancer Res. 53: 5244- 5250)), cell-surface antigen (TSTA) in a tumor-specific graft form ( Virus-induced tumor antigens, including envelope antigens of T-antigen DNA tumor virus and RNA tumor virus), neoplastic antigen-alpha-fetoprotein (e.g., CEA of the colon), bladder tumor neoplastic antigen (Hellstrom et al., 1985, Cancer Res. 45: 2210-2218), differentiation antigens (eg, human lung cancer (species) antigens L6, L20 (Hellstrom et al., 1986, Cancer Res. 46: 3917-3923), fibrosarcoma, human leukemia T cell antigen-Gp37 (Bhattacharya-Chatterjee et al., 1988, J. of Immun. 141: 1398-1403), glycoprotein, sphingolipids, breast cancer Antigens (e.g. EGFR (epithelial growth factor receptor)), HER2 antigen (p185 ^HER2 ), polymorphic endothelial mucin (PEM) (Hilkens et al., 1992, Trends in Bio. Chem. Sci. 17: 359), malignant human lymphocytes Antigen-APO-I (Bernhard et al., 1989, Science 245: 301-304), differentiation antigen (Feizi, 1985, Nature 314: 53-57) (e.g., I antigen found in neoplastic erythrocytes and primary endoderm), I (Ma) found in gastric adenocarcinoma, M18 and M39 found in breast endothelium, SSEA-1, VEP8, VEP9, Myl, VIM-D5 found in myeloid cells, D ₁ 56-22, TRA found in colorectal cancer -1-85 (blood group H), C14 found in colon adenocarcinoma (species), F3 found in lung adenocarcinoma (species), AH6 found in gastric cancer, Y hapten, Le ^y found in fetal cancer (species) cells , TL5 (blood group A), EGF found in A431 cells, pancreatic cancer The document found E ₁ series (blood group B), fetal cancer (species) that FC10.2, found in the stomach adenocarcinoma cells in adenocarcinoma (species) A CO-514 (blood group Le ^u) found in adenocarcinoma (kind of) Found NS-10, CO-43 (blood group Le ^b ), G49, EGF receptor (blood group ALe ^b / Le ^y ) found in colon adenocarcinoma (species), 19.9 found in colon cancer, gastric cancer-free, bone marrow cells Found T ₅ A ₇ , found in melanoma R ₂₄ , 4.2, G _D3 , D1.1, OFA-1, G _M2 , OFA-2, G _D2 , M1: 22 found in fetal carcinoma cells : 25: 8, SSEA-3 and SSEA-4 found in 4-8-cell stage embryos. In another embodiment, the antigen is a T cell receptor-derived peptide obtained from cutaneous T cell lymphoma (see Edelson, 1998, The Cancer Journal 4:62).

In another embodiment of the invention, the subject treated with the modified antibody of the invention is optionally treated with another cancer therapy such as surgery, radiation therapy or chemotherapy. In particular, the therapeutic agents of the invention used to prevent or treat cancer are administered in combination with a single chemotherapeutic agent or a compound of such a substance, examples of which are methotrexate, taxol, mercaptopurine, thio Guanine, urea hydroxide, cytarabine, cyclophosphamide, phosphamide, nitorourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, etoposide, camppatin, bleomycin, doxorubicin , Idarubicin, daunorubicin, dactinomycin, phyllomycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, pasililitaxel, docetaxel, and the like. It is not limited.

5.2.1.1. Malignant (species) cancer

Malignant cancers and other related diseases that can be treated or prevented by the administration of the present invention include those listed in Table 3, but are not limited to these (fishman et al., 1985, Medicine, 2d Ed., JB Lippincott Co. , Philadelphia)

TABLE 3

Malignant Cancer and Related Diseases

leukemia

Acute leukemia

Acute Lymphoblastic Leukemia

Acute myeloid leukemia

Myeloid

Myeloid

Myeloid

Construction

Red leukemia

Chronic leukemia

Chronic myeloid leukemia

Chronic Lymphocytic Leukemia

True erythrocytosis

Lymphoma

Hopkins disease

Non-Hopkins Disease

Multiple myeloma

Macroglobulinemia in Valentseng

H chain disease

Solid rock

Sarcoma and Cancer (species)

Fibrosarcoma

Myxedema

Liposarcoma

Chondrosarcoma

Osteosarcoma

Chuck

Vascular Sarcoma

Endothelial sarcoma

Lymphatic sarcoma

Lymphatic endothelial sarcoma

Live music

Mesothelioma

Ewing tumor

Smooth myoma

Colon cancer (species)

Pancreatic cancer

Breast cancer

Ovarian Cancer

Prostate cancer

Squamous cell carcinoma (species)

Basal cell carcinoma (species)

Adenocarcinoma (species)

Korean sarcoma

Blood Gland Sarcoma

Papillary carcinoma (species)

Papillary adenocarcinoma (species)

Cyst cancer (species)

Weeping Cancer (species)

Bronchial cancer (species)

Rectal Cell Carcinoma

Liver (cell) cancer

Cholangiocarcinoma (species)

Villi (species)

Normal somatoma

Born cancer (species)

Wilm Tumor

Cervical cancer

Uterine cancer

Testicular tumor

Lung cancer (species)

Small cell lung cancer (species)

Bladder Cancer

Epithelial cancer (species)

Glioma

Glioma

Medulloblastoma

Two pharynx

Celloma on Pinterest

Pineal carcinoma

Vestibular (cell) species

Auditory nerve bell

Rare <pip> dendrite

Meningioma

Melanoma

Neuroblastoma

Retinoblastoma

In certain embodiments, malignant cancer or dysplasia (metastasis and dysplasia), or hyperproliferative disease, is treated and prevented in the ovary, bladder, breast, colon, lung, skin, pancreas or uterus. In another embodiment, treating and preventing sarcoma, melanoma or leukemia.

5.2.1.2. Precancerous condition

The therapeutic agent of the present invention is administered to treat precancerous conditions and to prevent progression to neoplastic or malignant cancer conditions. Examples of such malignant cancers are shown in Table 3, but are not limited to these examples. This prophylactic or therapeutic use is prescribed for diseases known or suspected of developing neoplastic or cancerous progression, in particular when non-neoplastic cell growth consists of hyperplasia and dysplasia, especially when dysplasia occurs. (See Robbins and Angell, 197, Basic Pathology, 2d Ed., WB Saunders Co., Philadelphia, pp. 8-79 for a review of abnormal growth conditions). Hyperplasia is a controlled form of cell proliferation, including an increase in cell numbers in tissues or organs, with little change in structure or function. With one exception, endometrial hyperplasia often precedes endometrial cancer. Heterogeneity is a controlled form of cell growth in which one type of mature or fully differentiated cell is replaced with another type of mature cell. Hyperplasia can occur in endothelial or connective tissue cells. Common hyperplasia involves some disordered hyperepithelial cells. Dysplasia is a frequent precursor to cancer and is mainly found in the endothelium; This is the most disordered form of non-neoplastic cell growth, in which the unity of individual cells and the loss of organizational orientation of the cells occur. Dysplasia cells often have abnormally large, darkly colored nuclei and show polymorphism. Dysplasia occurs specifically in chronic redness or inflammation and is mainly found in the cervix, respiratory system, oral cavity and gallbladder.

In addition to the presence of abnormal cell growth characterized by hyperplasia, dysplasia or dysplasia, the presence of one or more features of the transformed phenotype or malignant phenotype manifested in vivo and in vitro by the patient's cell sample may prevent the composition of the vaccine. It is a measure of the need for treatment. As mentioned earlier, the characteristics of these transformed phenotypes include morphological changes, loose donation, loss of contact inhibition, loss of maintenance dependence, protease kdcnf, increased glycosylation, decreased serum need, expression of fetal antigens, 250,000. The disappearance of Dalton's cell surface proteins (see pp. 84-90, supra) for properties related to transformed or malignant phenotypes.

In certain embodiments, vitiligo, a benign-hyperplasia or dysplastic lesion of endothelial, or Bowin's disease (in situ carcinoma) is a pre-tumor lesion on which the need for preventive intervention is based.

In another embodiment, fibroblast disease (vesicle hyperplasia, breast dysplasia, certain adenomas (benign endothelial hyperplasia) are the criteria for the need for preventive intervention.

In another embodiment, a patient showing one or more source factors for malignant cancer is treated by administering an effective amount of a therapeutic drug of the present invention, which factors include chromosomal translocation associated with malignant cancer (eg, chronic osteosarcoma leukemia). For Philadelphia chromosome, t for follicular lymphoma (14; 18)), family-specific polyphosis or Gardner syndrome (precursor of colon cancer), benign monoclonal gambifaxi (precursion of multiple melanoma), Mendelian genetic pattern (E.g., family-specific colon cancer polyphosis, Gardner syndrome, genetic exostosis, polyendocrine adenomatosis, myeloid thymic carcinoma (amyloid) with amyloid production and chromaffin cell tumors, Poetz-Jay syndrome, Bon Reckling Ghausen's glioma, Retinoblastoma, Carotid carcinoma, Cutaneous melanoma (species), Intraretinal melanoma (species), Dry skin pigmentation, Ataxia Capillary dilatation, Cheddiac-Higashi syndrome, Cutaneous sclerosis, Lancony aplastic anemia, Blum syndrome) has a primary relationship with people with cancer or precancerous diseases (Robbins and Angell, 197, Basic Pathology, 2d Ed., WB Saunders Co., Philadelphia, pp. 112- 113).

In another specific embodiment, the therapeutic agent of the invention is administered to a human patient to prevent progression to cancer, melanoma or sarcoma of the ovary, breast, colon, lung, pancreas, skin, prostate, stomach, B lymphocytes or uterus do.

5.2.2. Treatment of Infectious Diseases

The invention also administers a therapeutic agent of the invention, in particular a modified immunoglobulin molecule (or a functionally active fragment, derivative or analog thereof, or a nucleic acid encoding a modified immunoglobulin, a functionally active fragment, analog or derivative thereof) By providing a method for preventing or treating an infectious disease, the immunoglobulin molecule is derived from an immunoglobulin molecule capable of immunospecific binding to an antigen of a pathogen causing an infectious disease or a cell receptor for an infectious disease pathogen. will be. As described below, infectious pathogens include, but are not limited to, viruses, bacteria, fungi, protozoa, and parasites.

In certain embodiments, the infectious disease comprises a method for preventing or treating an infectious disease by administering a modified immunoglobulin molecule (or functionally active fragment, derivative or analog thereof thereof, or a nucleic acid encoding a modified immunoglobulin). To provide, the immunoglobulin molecule is derived from an immunoglobulin that specifically recognizes one of the following infectious disease pathogens: Influenza virus hemagglutinin (Genbank accession no. J02132; Air, 1981, Proc. Natl.Acad. Sci. USA 78: 739-743; Newton et al., 1983, Virology 128: 495-501), human respiratory syncolitic virus G glycoprotein (Genbank accessionno. Z33429; Garcia et al., 1994, J Virol .; Collins et al., 1984, Proc. Natl. Acad. Sci. USA 81: 783), deep proteins, parent proteins or other proteins of the dengue virus (Genbank accession no. M19197; Hahn et al., 1988, Virology 12: 17-180), measles bar Irus hemagglutinin (Genbank accession no. M81899; Rota et al., 1992, Virology 188: 135-142), herpes zoster 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 (eg, gp120, Putney et al., 198, Science 234: 1392-1395) , Hepatitis B surface antigen (Itoh et al., 198, Nature 308: 19; Neurath et al., 198, Vaccine 4:34), Diphtheria toxin (Audibert et al., 1981, Nature 289: 543), Streptococcus Streptococcus 24M epitooff (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 ) Viral glycoprotein H, Pseudorabies virus glycoprotein gIII (gpC), spreadable gastroenteritis glycoprotein 195, spreadable gastroenteritis parent protein, porcine rotavirus glycoprotein 38, porcine parvovirus capsid protein, sulfurina Serpulina hydodysenteriae protective antigen, bovine viral diarrhea glycoprotein 55, Newcastle disease virus hemagglutinin-neuuraminidase, swine cold hemagglutinin, swine cold nuuraminidase, foot-and-mouth virus, swine Cholera virus, swine influenza virus, African swine fever virus, Mycoplasma hypopnemoniae, infectious bovine nasal bronchitis virus (eg, infectious bovine nasal cavity) Gitis protein E or glycoprotein G), infectious laryngitis virus glycoprotein G or glycoprotein I), glycoprotein of La Crosse virus (Gonzales-scarano et al., 1982, virology 120: 42), fetal diarrhea virus (Matsuno and Inouye, 1983, Infection and Immunity 39: 155), Venezuelan equine encephalomyelitis virus (Mathews and Roehrig, 1982, J. Immunol. 129: 273), Punta Toro virus (Dalrymple et al., 1981, in Replication of Negative Strand Viruses, Bishop and Compans (eds.), Elsevier, NY, p. 17), murine leukemia virus (Steeves et al., 1974, J. Virol. 14: 187 ), Mouse breast tumor virus (Massey and Schochetman, 1981, Virology 115: 20), hepatitis B deep protein or hepatitis B surface antigen (UKPatent Publication No. GB 2034323A published June 4, 1980; Ganerm and Varmus, 1987, Ann, Rev. Biochem. 5: 51-93; Tiollais et al., 1985, Nature 317: 489-495), antigens of Equine Influenza Virus or Equine Herpes Virus (e.g., Type A (Equine Influenza Virus / Alaska 91 New Uramidinase, Equine Influenza Virus Type A / Miami 63) Neuuraminidase, Equine Influenza Virus Type A / Kentucky 81 Neuuraminidase, Equine Herpesvirus Type 1 Glycoprotein B, Equine Herpesvirus Type 1 Glycoprotein D), Bovine Respiratory Synthetic Virus or Bovine Parainfluenza Virus (e.g., Bovine respiratory syncytial virus adherent protein (BRSV G), bovine respiratory syncytotic virus fusion protein (BRSV F), bovine respiratory syncytial virus nucleocapsid protein (BRSV N), bovine parainfluenza virus type 3 fusion protein, bovine parainfluenza Virus type 3 hemagglutinin nuuraminidase), bovine viral diarrhea virus glycoprotein 48, bovine diarrhea virus sugar White matter 53. In another specific embodiment, the infectious disease is treated or prevented by administering a modified immunoglobulin (or a functionally active fragment, derivative or analog thereof, or a nucleic acid encoding them), wherein the modified immunoglobulin is associated with a corresponding protein. Pathogens and cellular receptors are used to recognize cellular receptors for infectious disease etiology, a list of pathogens and cellular receptors is presented in Table 4.

Table 4

Viral diseases that can be treated or prevented by the methods of the present invention include hepatitis A, hepatitis B, hepatitis C, influenza, chickenpox, adenovirus, herpes simplex virus type I (HSV-I), and herpes simplex type II (HSV-I). ), Erosion, nasal cold virus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, ecino virus, arbovirus, hantan virus, coxsackie virus, mumps virus, measles Virus, rubella virus, poliovirus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-I), picornavirus, enterovirus, calciviridae, nord Walk virus group, togavirus (e.g., dung virus), alphavirus, flavivirus, coronavirus, labyze virus, marburg virus, Bolavirus, parainfluenza virus, orthomyxovirus, field virus, arenavirus, leovirus, rotavirus, orbivirus, human T cell leukemia virus type I, human T cell leukemia virus type II, apes immunodeficiency virus, lentivirus, Diseases caused by poloomavirus, parvovirus, Epstein-Barr virus, human herpesvirus-6, monkey herpesvirus 1, faxvirus, encephalitis are included, but are not limited to these.

Bacterial diseases that can be treated or prevented by the methods of the present invention include Gram-negative and Gram-positive bacteria, Mycobateria rickettsia, Mycoplasma, Neisseria species (eg, Neisseria mennigitidis, neisseria gonorrhoeae), Legionella ( legionella, Shigella spp., Vibrio cholerae, Streptococcus pneumoniae, Cornebacteria diphtheriae, Clostridium tetani, Bordetella perlella Syphilis induced or caused by bacteria, including but not limited to, Bossetella pertussis, Haemophilus spp., Chlamydia spp., Intestinal toxin Escherichia coli Or Lyme disease and bacterial disease.

Protozoan diseases that can be prevented or treated by the methods of the present invention are caused by protozoa, including but not limited to malaria, eymeria, resimania, coccidioa, trimansomoma, mold (eg, Candida). .

In certain embodiments of the invention, the therapeutic agents of the invention are administered in combination with a suitable antibiotic, anti-fungal, anti-viral or other drug useful for the treatment or prevention of the disease of the infection.

5.3. Gene therapy

Gene therapy refers to the prevention or treatment of a disease by administering a nucleic acid to a subject having a disease associated with the expression of an antigen, which antigen is recognized by an immunoglobulin from which the modified globulin molecule is derived. For example, a disease or condition is an cancer associated with the expression of a specific cancer or tumor antigen, or an infectious disease associated with the expression of a specific antigen of an infectious disease or an infectious disease pathogen in combination with a specific cellular receptor. In certain embodiments of the invention, the therapeutic nucleic acid encodes a sequence that produces an intracellular (without lead sequence) or intracellular (with lead sequence) modified immunoglobulin.

For a general review of gene therapy methods, see Goldspiel et al., 1993, Clinical Pharmacy 12: 488-505; Wu and Wu 1991, Biocherapy 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. See 62: 191-217. Methods generally known in the recombinant DNA art that can be used are described in Ausubel et al., 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.

In another aspect, the therapeutic nucleic acid comprises an expression vector expressing a modified immunoglobulin molecule.

The delivery of the nucleic acid to the patient may be direct, in which case the patient may be directly exposed to the nucleic acid or nucleic acid-delivery vector, or delivery complex, and indirect, in which case the cell is first transformed with the nucleic acid in vitro. And then transplanted into the patient. Each of these two approaches is known in vivo or ex vivo gene therapy.

In certain embodiments, the nucleic acid is administered in an integrated body, in which case it is expressed to produce an antibody. This can be accomplished by any of a number of methods known in the art, for example, by constructing it as part of a suitable nucleic acid expression vector and administering it so that it is placed in the cell, thereby causing a defective or attenuated retrovirus or other Using viral vectors (see US Pat. No. 4,980,286), direct injection of naked DNA, microparticle explosion (e.g., Genel: Biolistic, Dupont), into lipids, cell-surface receptors or transfection drugs Coating to encapsulate in the form of a biopolymer (e.g. poly-β-1-> 4-N-acetylglocosamine polysaccharide; see US Patent No. 5,635,493), encapsulation in the form of liposomes, microparticles, microcapsules, nuclei It is administered by linking it to a known peptide or by linking it to a target ligand of receptor-mediated endocytosis (Wu and Wu, 1987, J. Biol. Chem. 262: 442). 9-4432). In another embodiment, nucleic acid-ligand complexes can be made, wherein the ligands comprise fusion viral peptides that perturb endosomes to allow nucleic acids to avoid liposome degradation. In another embodiment, nucleic acids can be targeted in vivo for cell specific adsorption and expression using specific receptors (PCT Publications WO92 / 06180 dated April 16, 1992 (Wu et al.); WO 92/22635 dated December 23, 1992 (Wilson et al.); WO 92/20316 dated November 26, 1992 (Findeis et al.); WO 93/14188 dated July 22, 1993 (Young). Alternatively, nucleic acids can be introduced into cells and inserted between 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.

Alternatively, single chain antibodies such as neutralizing antibodies that bind to intracellular epitooff can also be administered. Such single chain antibodies can be administered by expressing nucleotide sequences encoding single chain antibodies in a target cell population using techniques known in the art (Marasco et al., 1993, Proc. Natl. Acad. Sci. USA 90: 7889-7893

Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3: 499-503). Adenoviruses are other viral vectors that can be used for gene therapy. Adenoviruses are particularly attractive carriers for gene transfer to respiratory epithelia, which cause minor diseases in the respiratory epithelium. Other targets for the adenovirus-based delivery system are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson (1993, Current Opinion in Genetics and Development 3: 499-503) provide an overview of adenovirus-based gene therapy. Bout et al. (1994, Human Gene Therapy 5: 3-10) show how to transfer genes to respiratory epithelia in rhesus monkeys. Other examples of use of adenoviruses in gene therapy include Rosenfeld et al., 1991, Science 252: 432-434; Rosenfeld et al., 1992, Cell 68: 143-155; Mastrangeli et al., 1993, J. Clin. Invest. Found at 91: 225-234. Adeno-associated virus (AAV) is also being considered for use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med 204: 289-300).

The type and amount of therapeutic nucleic acid for use depends on the type of disease, the degree of effect desired, the patient's condition, and can be determined by one skilled in the art.

5.3. Vaccine Compositions and Administration

The present invention also provides a vaccine composition containing the therapeutic drug of the present invention, wherein the vaccine composition is suitable for administration to induce a protective immune (bodily and / or cell mediated) response to a specific antigen.

Suitable formulations of such vaccines include injectables (liquid solutions or suspensions); It may also be in solid form suitable for dissolution or suspension prior to injection. The formulation is emulsified or the peptide is encapsulated in liposome form. Active immunogenic ingredients are often mixed with excipients, which are pharmaceutically acceptable and complementary to the active ingredient. Suitable carriers include water, saline, buffered saline, glucose, glycerol, ethanol, sterile isotonic water soluble buffers, compounds thereof. In addition, if desired, vaccine formulations also include small amounts of additives, such as wetting or tanning agents, pH relaxants, anti-antibodies that increase the efficacy of the vaccine.

Examples of effective anti-antigens include aluminum hydroxide, N-acetyl-mural-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramil-L-alanyl-D-isoglutamine , N-acetylmurayl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1′-2′-dipalmitoyl-nu-glycero-3-hydroxyphosphoryloxy)- Although ethylamine is mentioned, It is not limited to these examples.

The efficacy of an antiantibody is determined by measuring the induction of anti-genetic antibodies against injected immunoglobulins made of specific antiantibodies.

The composition may be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation or powder. Oral compositions may include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium, stearate, sodium saccharide, cellulose, magnesium carbonate.

Generally, the ingredients are provided individually or in admixture in unit dosage form, the form of which is a lyophilized powder or anhydrous concentrate on a sealed container, an example of which is an ampoule or yarn indicating the amount of active ingredient. Kits. When the composition is administered by injection, an ampoule of sterile diluent is provided to mix the components before administration.

In certain embodiments, the lyophilized modified immunoglobulin of the present invention is provided in a first container; The second container contains a diluent consisting of an aqueous solution of 50% glycerin, 0.25% phenol and a preservative (eg 0.005% brilliant green).

The invention also provides pharmaceutical packs or kits, which consist of one or more containers filled with one or more of the vaccine composition components of the invention. Such containers follow the guidelines of the governmental authority on controlling the manufacture, use, or sale of pharmaceuticals or biological products, and must be approved by the authority responsible for manufacturing, use, or sale when administered to humans.

If desired, the compositions are provided in packs or vending machines, which contain one or multiple unit doses containing the active ingredient. The pack consists of a piece of metal or plastic, such as a blister pack. Picks or vending machines follow the instructions for administration. Compositions comprising the compounds of the invention in the form of complementary pharmaceutical carriers are also prepared, placed in suitable containers, and labeled for the indicated disease treatment.

The individual receiving the vaccine is preferably a mammal, most suitably a human, but may be other than human, for example cattle, horses, sheep, pigs, chickens (eg chicks), goats, cats, puppies, hamsters. , Mice or rats, but not limited to these examples.

Many methods can be used to introduce a vaccine composition of the present invention; Examples of such methods include oral, cerebral, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal routes, bleeding (scratching the top layer of skin using a bifurcated needle), or any other immunization route. Although it may be, it is not limited to these examples. In certain embodiments, bleeding is used.

The exact daily dose of the modified immunoglobulin molecule used in the form of the composition depends on the route of administration and the condition of the patient, which is determined in consideration of the condition of the physician and each patient according to standard clinical techniques. The effective amount of immunity is an amount sufficient to elicit an immune response against the modified immunoglobulin in the host to which the vaccine is administered. Effective daily doses may be estimated from dose-response curves obtained from animal model testing apparatus.

5.4. How to produce modified immunoglobulins.

The modified immunoglobulins of the present invention can be made by any method known in the art for immunoglobulin synthesis, and in particular by synthetic or recombinant expression, preferably by recombinant expression techniques.

Recombinant expression of the modified immunoglobulins, fragments, analogs or derivatives thereof of the present invention requires the construction of nucleic acids encoding modified immunoglobulins. If the nucleotide sequence of the modified immunoglobulin is known, then the nucleic acid encoding the modified immunoglobulin is synthesized from chemically synthesized oligonucleotides (Kutmeier et al., 1994, BioTechniques 17: 242), in brief hereby referred to as modified immunoglobulin. Synthesis of overlapping oligonucleotides having portions of the sequence encoding the globulin, annealing and ligation of these oligonucleotides, and then amplifying the ligation oligonucleotides by PCR.

Alternatively, the nucleic acid encoding the modified immunoglobulin is made from the nucleic acid encoding the immunoglobulin derived from the modified immunoglobulin. Clones bearing nucleic acids encoding specific immunoglobulins cannot be obtained, but if immunoglobulin molecular sequences are known, nucleic acids encoding immunoglobulins may be derived from a suitable source (antibody cDNA library or any tissue or cell expressing immunoglobulin). CDNA library) can be obtained using PCR amplification using synthetic primers that hybridize with the 3 'and 5' ends of the sequence or hybridization methods using oligonucleotide probes specific for a specific gene sequence.

If an immunoglobulin molecule that specifically recognizes a particular antigen is not available (or there is no cDNA library source for cloning a nucleic acid encoding such immunoglobulin), immunoglobulins specific for a particular antigen are known in the art. Methods, for example, immunization of animals such as rabbits, polyclonal antibodies, and more preferably polyclonal antibodies (Kohler and Milstein, 1975, Nature 256: 495-497). Kozbon et al., 1983, Immunology Today 4:72; Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) .Alternatively, at least the immunoglobulin Clones encoding the Fab moiety can be selected by selecting Fab expression libraries for clones of Fab fragments that bind to specific antigens (Huse et al., 1989, Science 246: 1275-1281) or by selecting antibody libraries (Clackson et al). ., 1991, Nature 352: 62 4; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94: 4937.

Once the nucleic acid encoded at least the variant domain of immunoglobulin was obtained. Any suitable cloning vector is introduced and a region of the immunoglobulin molecule is introduced into a vector containing encoded nucleotides (PCT Publication WO 86/05807; PCT Publication WO 89/01036; US Patent No. 5,122,464; and Bebbington , 1991, Methods in Enzymology 2: 136-145. Vectors containing complete L or H chains for co-expression with nucleic acids that allow expression of complete antibody molecules are also available. The nucleic acid encoding the immunoglobulin is then modified to introduce nucleotide substitutions or deletions, which, together with substitutions, deletions or insertions of any other desired amino acids, SH one or multiple variant region cysteine residues that participate in disulfide bonds between the chains. It is necessary to substitute with an amino acid residue having no group. Such modifications can be carried out by any method known in the art for the introduction of specific mutations or deletions in nucleotides, such as chemical mutations, in vitro mutagenesis (Hutchinson et al., 1979, J. Biol. Chem. 253: 6551) and PCR-based methods, but not limited to these examples.

In addition, a technique for producing chimeric antibodies by combining genes obtained from human antibody molecules with appropriate bioactivity with genes derived from mouse antibody molecules having appropriate antigen specificity (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. As described above, chimeric antibodies are molecules derived from different species of animals, for example, murine monoclonal antibody-derived mutation regions and human immunoglobulins derived from certain regions (e.g., antibodies adapted to the human body). It can be mentioned.

Alternatively, techniques for the production of single chain antibodies (US 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 modified to produce single chain antibodies. Single chain antibodies are made by connecting the H and L chains of the Fv region through an amino acid bridge, thereby producing a single chain polypeptide. Combination techniques of functional Fv fragments in E. coli can also be used (Skerra et al., 1988, Science 242: 1038-1041).

Antibody fragments that recognize specific epitopes are made by known techniques. For example, such fragments include, but are not limited to, F (ab ') ₂ fragments, Fab fragments made by reducing disulfide bonds of F (ab') ₂ fragments, which can be made by pepsin cleavage of antibody molecules.

Once the nucleic acid encoding the modified immunoglobulin molecule of the present invention is obtained, the vector for producing immunoglobulin molecule is made by recombinant DNA technology using techniques known in the art. The modified immunoglobulin molecule is then recombinantly expressed and isolated by any method known in the art (eg, the method identified in section 6) (see Bebbington, 1991, Methods in Enzymology 2: 136-145,). In brief, COS cells or any other suitable cultured cell are transiently or non-temporarily transfected with an expression vector encoding modified immunoglobulin, incubated for a suitable period of time to express immunoglobulin, and then the supernatant is COS The cells are harvested, wherein the supernatant retains secreted, modified immunoglobulins.

Methods known to those skilled in the art can be used to construct expression vectors carrying immunoglobulin molecular coding sequences, appropriate transcriptional and translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. 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, John Wiley & Sons, NY See the description given by).

Expression vectors are transferred to host cells by conventional techniques, and the transfected cells are cultured by conventional methods to produce the immunoglobulins of the present invention.

The host cell used for expressing the recombinant antibody of the present invention is a bacterial cell such as Escherichia coli, or preferably a eukaryotic cell, particularly a cell for the expression of the whole recombinant immunoglobulin molecule. In particular, mammalian cells, such as Chinese hamster ovary cells (CHO), bind to vectors such as the major intermediate early gene promoter factor of human cytomegalovirus, which is a useful expression system for immunoglobulins (Foecking et al., 198, Gene 45: 101; Cockett et al., 1990, Bio / Technology 8: 2).

Various host-expression vector systems are used to express the modified immunoglobulin molecules of the present invention. Such host-expression systems exhibit carriers that produce and subsequently purify the coding sequence of interest, but also express the immunoglobulin molecule of the invention in situ when transformed or transfected with the appropriate nucleotide coding sequence. Also indicates. These include bacteria (E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors carrying an immunoglobulin coding sequence; Yeast (Saccharomyces, Pichia) transformed by a recombinant yeast expression vector carrying an immunoglobulin coding sequence; Insect cells infected with a recombinant virus expression vector (eg, baculovirus) having an immunoglobulin coding sequence; Plant cell lines infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or a recombinant plasmid expression vector (e.g. Ti plasmid) carrying an immunoglobulin coding sequence; Or a mammal having a recombinant expression construct with a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus post-promoter, a duplex virus 7.5K promoter) Cell lines (eg, COS, CHO, BHK, 293, 3T3 cells).

In bacterial systems, a number of expression vectors are preferentially selected depending on the intended use for the immunoglobulin molecule to be expressed. For example, when producing such proteins in large quantities for the production of pharmaceutical compositions of immunoglobulin molecules, vectors that control the high level expression of fusion protein products that are readily purified are desirable. Such vectors include Escherichia coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2: 1791), wherein the immunoglobulin coding sequence is individually ligated into a vector frame through the lac Z coding region and is a fusion protein. This is made: PIN vector (Inouye & Inouye, 1985, Nucleic Acids Res. 13: 3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24: 5503-5509. It is expressed as a fusion protein with S-transferase (GST) In general, these fusion proteins are water soluble and can be easily purified from lysed cells, which absorb and bind to matrix glutathione-agarose beads Elution is carried out in the presence of free glutathione The pGEX vector is designed to contain a thrombin or factor Xa protease cleavage so that the cloned target gene product falls from the GST moiety.

In the insect system, Autographa californica nuclear polyhedron virus (AcNPV) is used as a vector to express foreign genes. The virus is grown in Spodoptera frugiperda. Immunoglobulin coding sequences are individually cloned into non-essential sites of the virus and placed under the control of an AcNPV promoter (eg, polyhedral promoter).

In mammalian cells, a number of virus-based expression systems are used. When adenovirus is used as the expression vector, the immunoglobulin coding sequence of interest is ligated to the adenovirus transcriptional / detoxification control complex (e.g. post-promoter, trilateral lead sequence). This chimeric gene is subsequently inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into non-essential regions (eg, E1 or E2 regions) of the viral genome results in recombinant viruses that survive and are capable of expressing immunoglobulin molecules in infected hosts (Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81: 355-359). Certain initiation signals are required for efficient translation of inserted immunoglobulin coding sequences. These signals include ATG start codons and contiguous sequences. Initiation codons also keep pace with the translation framework of the desired coding sequence so that the entire insert is translated. These exogenous detox control signals and initiation codons can be obtained from various sources (natural, artificial). The efficiency of expression can be enhanced by the insertion of appropriate transcription enhancers, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153: 51-544).

In addition, the host cell line is selected to express the inserted sequence and to modify and regulate the gene product in the specific format desired. Such modifications (eg, sugar additions) and processing (eg, cleavage) of protein products are important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational process and for modification of proteins and gene products. Appropriate cell or host systems are selected to allow for the correct modification and processing of foreign expression proteins. For this purpose, eukaryotic host cells with appropriate processing, sugar addition, and cellular mechanisms for phosphorylation of gene products are used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38.

For long-term, high-yield production of recombinant proteins, proper expression is preferred. For example, cell systems that stably express immunoglobulin molecules are processed. Rather than using expression vectors with replication of viral origin, host cells can be transformed with DNA regulated with appropriate expression regulators (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectivity marks. have. After introduction of the foreign DNA, the processed cells are grown 1-2 days in complete medium and then transferred to selective medium. Selectivity marks on the recombinant plasmids provide resistance to selectivity and allow cells to stably integrate the plasmid into the chromosomes to form lesions, which can then be cloned and expanded into the cell line. This method is used preferentially to process cell lines expressing immunoglobulin molecules. This processed cell line is particularly useful for the selection and evaluation of compounds that interact directly or indirectly with immunoglobulin molecules.

Multiple screening systems are used, examples of which are herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11: 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. NA Acad. Sci. USA 48: 202), adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) genes can be used for tk ⁻ , hgprt ⁻ or aprt ⁻ cells, respectively. In addition, metabolite resistance can be used as the basis for selection of the following genes: dhfr (Wigler et al., 1980, Natl. Acad. Sci. USA 77: 357; O, when conferring resistance to methotrexate). Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1527); Gpt (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78: 2072) when conferring resistance to mycofinolic acid; When conferring resistance to aminoglycoside G-418, neo (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 Morgan and Anderson, 1993, Ann, Rev. Biochem. 62: 191-217; May, 1993, TIBTECH 11 (5): 155-215). Methods known in the recombinant DNA technology that can be used are described in Ausubel et al. (Eds.), 1993, Current Protocols in Molecular Bioloby, 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. Presented at 150: 1.

Alternatively, any fusion protein is readily purified using antibodies specific for the fusion protein expressed. For example, Janknecht et al. Can readily purify non-denatured fusion proteins expressed in human cell systems (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88: 8972-897). In this system, the gene of interest is so cloned with the vaccinia recombinant plasmid such that the open translation frame of the gene is fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a binding domain to the fusion protein. Extracts obtained from cells infected with recombinant vaccinia virus were loaded onto a Ni ²⁺ nitriloacetic acid-agarose column and the histidine-tag protein was eluted selectively with imidazole-containing buffer.

Expression levels of immunoglobulin molecules can be increased by vector amplification (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 the mark on the vector line expressing an immunoglobulin is amplifiable, the number of copies of the mark gene is increased due to the increase in the level of inhibitor present in the culture of the host cell. Since the amplified region is associated with an immunoglobulin gene, the production of immunoglobulin also increases (Crouse et al., 1983, Mol. Cell. Biol. 3: 257).

The host cell is cotransfected with two expression vectors of the invention, a first vector encoding H chain induction polypeptide and a second sequence encoding L chain induction polypeptide. Both vectors carry the same selectivity mark, which expresses equally H and L chain polypeptides. Alternatively, a single vector encoding both H and L chain polypeptides is used. In this case, the L chain is located in front of the H chain to avoid excessive increases in toxic free H chains (Proudfoot, 1986, Nature 322: 52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77: 2197). . Coding sequences for H and L chains consist of cDNA or genomic DNA.

Once the modified immunoglobulins of the present invention are recombinantly expressed, they are purified by any method known in the art for immunoglobulin purification, which involves chromatography (e.g., ion exchange, affinity, in particular similar to protein A). Affinity for the antigen, column size chromatography), centrifugation, solubility difference or any other standard technique for protein purification.

5.5. Demonstration of therapeutic use

Modified antibodies of the present invention can be selected and analyzed with various efficacy in the treatment or prevention of specific diseases.

First, the immune capacity of the vaccine composition containing the modified antibody of the present invention can be determined by monitoring the anti-genetic response of the experimental animals after vaccine immunization. The development of a humoral response is taken as a sign of a systemic immune response, the other of which is important for protection from disease, especially cell-mediated immunity. Experimental animals include mice, rabbits, chimpanzees and, ultimately, human subjects. The vaccine made in the present invention can be experimentally made to infect chimpanzees. However, because chimpanzees are protected animal species, the antibody response to the vaccines of the present invention is first described in order to find one or more optimal immunoglobulin molecules or optimal compounds for immunoglobulin molecules for use in chimpanzee efficacy studies. Can study on animals.

The immune response of the test subject is known against antibodies by using known techniques, such as enzyme-immunoassay (ELISA), immunoblot, radioimmunoprecipitation, or attenuation of disease symptoms in protected or immunized porridge. Analyze in a variety of ways, such as the reactivity of the generated immune serum.

As an example of a suitable animal experiment, the vaccine composition of the present invention is tested in rabbits for the ability to induce anti-genetic responses to modified immunoglobulin molecules. For example, male specific-pathogen-free (SPF) young New Zealand white adult rabbits are used. The rabbit experimental group each ingested an effective amount of vaccine. The rabbit control group is injected with 1 mM Tris-HCl (pH 9.0) of a vaccine containing naturally occurring antibodies. Blood samples are taken from rabbits on a weekly or two-week basis, and serum is derived from anti-genetic antibodies against modified immunoglobulins and anti-antibodies specific for antigens directly directed to the modified antibodies using Abbort Laboratories. Analyze genotype antibodies The presence of anti-genetic antibodies is analyzed using ELISA. Because rabbits have a variety of responses due to their non-negative nature, it is useful to test vaccines in mice.

In addition, the modified antibodies of the present invention are first administered to a test subject (animal or human), and then an anti-anti-genetic antibody (ie, an Ab3 antibody) made as part of an anti-genetic response to the injected modified antibody. Test separately. The isolated Ab3 was then tested for binding to specific antigens (eg tumor antigens, antigens of infectious diseases) by any immunoassay known in the art. Examples of such immunoassays include radioimmunoassay, ELISA, " Sandwich "immunoassay, gel diffusion precipitation reaction, immunodiffusion analysis, western blot, sedimentation reaction, aggregation reaction analysis, complement fixation analysis, immunofluorescence analysis, protein A analysis, immunoelectrophoresis analysis, but not limited to these examples Do not.

When the modified antibody is administered directly to a cancer or a tumor, the therapeutic effect of the isolated Ab3 against cancer, tumor or other neoplastic disease may be achieved by culturing the cancer or tumor from the patient, contacting the cells with the Ab3 antibody to be tested, Ab3 The proliferation or growth of cells in contact with the antibody is selected by comparing the proliferation or growth of cells not in contact with the Ab3 antibody, wherein much lower levels of proliferation or growth of the cells in contact are selected from the Ab3 antibody (modified antibody immunization of the present invention). Induced) is effective for treating cancer in patients. Many standard assays in the art have been used to assess this survival or growth; For example, cell proliferation

Assessment of ³ H-thymidine insertions, direct cell counts, detection of changes in transcriptional activity of known genes such as proto-tumor genes, or cell cycle marks; Cell survival can be assessed by trypan blue staining and differentiation can be assessed based on morphological changes. If the modified antibody is directly directed against the antigen of the infectious disease agent, the isolated Ab3 is tested for activity in any in vitro activity test for the particular pathogen.

In addition, the modified antibodies of the present invention are tested directly in vivo. In order to monitor the effects of the therapeutic agents of the present invention, the level of antigen targeted by the modified antibody is measured at appropriate time intervals before, during and after treatment. Changes or absence of changes in antigenic amounts can be identified and associated with therapeutic effects on the subject.

In particular, in the case of cancer treatment, the serum level of the antigen has a direct correlation with the degree of cancer (e.g. breast cancer), poor prognosis. In general, reduction of antigen levels is associated with effective treatment.

If 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 antigenic level of the infectious disease agent at appropriate time intervals before, during and after treatment, wherein It suggests that the reduced modifying antigen is effective.

In an appropriate aspect, a method that can be taken is to determine the levels of antigen at different time points and compare these levels with the basal level. The basal level will be the mark level present in normal disease-free individuals; Pre-treatment level, during disease improvement or during stability. These levels can be correlated with disease course or treatment outcome.

Antigen levels can be determined by any method known in the art. For example, specific antigens can be quantified by known immunodiagnostic methods, such as Western blotting immunoprecipitation, in which methods use antibodies to specific antigens.

The intensity of the in vivo immune response to modified immunoglobulins is determined by any method known in the art, including retarded ultrasensitive skin testing, in vitro activity analysis of cell fusion T-lymphocytes. It is not limited.

Retarded supersensitive skin testing is of great importance in experiments with overall immunity to antigen and cellular immunity. Appropriate techniques in skin testing require antigen sterilization at 4 ° C, light blocking, and reconstitution immediately before use. The antigen is administered transdermally rather than subcutaneously using a 25- or 27-gauge needle. 24 hours, 48 hours after transdermal administration of antigen, the largest erythema and sclerosis are measured by rule. Low activity against any antigen or group of antigens is confirmed by repetition of higher concentrations of antigen or intermediate experiments in a similar environment.

To test for cytolytic T-lymphocyte activity, T-lymphocytes isolated from immunized subjects using centrifugal concentration difference techniques were tested for antigens targeted by the modified antibody in 3 ml PRMI medium containing 10% fetal bovine serum. Restimulate with retained cells. In some experiments 33% secondary hybrid lymphocyte culture supernatants or IL-2 are included in the culture medium as T cell growth factors. To measure the primary response of cytolytic T-lymphocytes after immunization, isolated T cells are cultured with and without antigen. After 6 days, the cultures are tested for cytotoxicity in a 4 hour ⁵¹ Cr-release assay. ⁵¹ Cr-release of spontaneous targets is below 20% when immunization is effective (Heike et al., J. Immunotherapy 15: 15-174).

In another aspect, modified immunoglobulins test efficacy by monitoring a subject for improvement or recovery from a particular disease or condition associated with the antigen to which the modified antibody is targeted. When the modified antibody targets a tumor or cancer antigen, the progress of the particular tumor or cancer is tracked by any known diagnostic or screening method for monitoring the cancer or tumor. For example, cancer or tumor progression is monitored by analyzing the level of a particular cancer or tumor antigen (or another antigen associated with a particular tumor or cancer) in the serum of an individual or by injecting labeled antibodies specific for the antigen. Other imaging techniques, such as computed tomography (CT) scans or sonographs, are also used to monitor cancer or tumor progress. Biopsies are also performed. Prior to conducting this test in humans, potency testing of modified immunoglobulins may be performed in animal models of specific cancers or tumors.

In the case of an infectious disease, the efficacy of the modified antibody can be analyzed by administering the modified antibody to a subject (human subject or animal disease model) and then monitoring the symptoms of the specific infectious disease etiology or specific infectious disease. The level of infectious etiology is determined by any method known in the art for the analysis of infectious etiology, in the case of viral or bacterial levels, for example viral titers. The level of infectious disease etiology is also determined by measuring the antigen that the modified immunoglobulin targets or another antigenic level of infectious disease etiology. Reducing the level of infectious disease or reducing the symptoms of infectious disease suggests that the modified antibody is effective.

6. EXAMPLES: Anti-Genetic Vaccine Inducers for Colon Cancer

This example describes the construction of a modified antibody derived from the monoclonal antibody MAb31.1 (Hybridoma Secretion Mab31.1 is available from American Type Tissue Collection as accession No. HB12314). Mab31.1 recognizes antigens expressed by Sarah colon cancer (species). The modified antibody of the present invention based on Mab31.1 is processed to replace the cysteine residues of the H and L chain variant regions with alanine. Thus, the resultant modified antibody lacks interchain disulfide bonds in H and L chain variant regions.

6.1. Construction of Modified Antibodies

A method of constructing a modified antibody consists of constructing two processing genes encoding H and L chain mutation regions, wherein a particular cysteine residue known to be important for disulfide bonds between chains is modified with alanine. Alanine residues are those substituted with cysteine residues at positions 22 and 92 of the H chain mutation region of the antibody derived from Mab31.1 or cysteine residues at positions 23 and 88 of the L chain mutation region of the antibody derived from Mab31.1. To construct these processing genes, oligonucleotide groups were combined (as described below) and inserted into a suitable vector providing a region.

In order to construct a mutation region gene encoding CDRs without disulfide bonds between chains, the following procedure was carried out.

First, the first oligonucleotide is annealed to form a sticky double-stranded DNA fragment (shown in FIG. 10, step 1). In particular, oligonucleotides of about 80 base lengths corresponding to sequences having 20 base overlap regions were synthesized using GenoSys Biotech Inc.'s automated technology. Specific sequences of each of these oligonucleotides are shown in FIGS. 9A and 9B. Fig. 9A shows a list of ten oligo groups used for processing the H chain variant region gene called 2CAVHCOL1. 2CAVHCOL1 lacks two cysteine residues when compared to a common H chain variant gene. 9B shows a list of twelve oligo groups used to process the L-chain variant region gene called 2CAVLCOL1. 2CAVLCOL1 lacks two cysteine residues when compared to the consensus L chain variant gene. In order to bind oligos with desired genes, 10-12 oligo groups were combined as described below and presented in FIG. 10, wherein the nature of 1-10 oligos indicated in FIG. 10 is shown in Table 5. Prior to binding, each oligonucleotide was 5 ′ phosphorylated as follows: 25 μl of each oligo was incubated for 1 hour at 37 ° C. in the presence of T4 polynucleotide kinase, 50 mM ATP. The reaction was stopped by heating at 70 ° C. for 5 minutes and then ethanol precipitated. Once phosphorylated complementary oligonucleotides (oligo 1 + oligo 10, oligo 2 + oligo 9, oligo 3 + oligo 8, oligo 4 + oligo 7, oligo 5 + oligo 6) (Figure 10) are then mixed in a sterile centrifuge tube and The tube is annealed by heating in a water bath at 65 ° C. for 5 minutes and then cooled at room temperature for 30 minutes. When annealed, short two-stranded DNA fragments with sticky ends are produced.

The sticky two-stranded DNA fragments are then ligated into longer strands until the combined mutagen genes are combined (Figure 10, steps 2-4). In particular, sticky two-stranded DNA fragments were ligated in a water bath maintained at 16 ° C. for 2 hours in the presence of T4 DNA ligase, 10 mM ATP. The annealed oligo 1/10 was mixed with the annealed oligo 2/9 and the annealed oligo 3/8 with the annealed oligo 4/7. The resulting oligo was recorded as oligo 1/10/2/9 and oligo 3/8/4/7. Oligo 3/8/4/7 was then ligated to oligo 5/6. The resulting oligo 3/8/4/7/5/6 was then ligated with oligo 1/10/2/9 to produce full length mutation regions genes.

Alternatively, when using 12 oligo groups, the order of addition is as follows: 1 + 12 = 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 = 1/12/1/11, 3/10 + 4/9 = 3/10/4/9, 5/8 + 6/7 = 5/8/6/7, 1/122/11 + 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 variant region gene. The oligonucleotide names used in the construction of the process genes are shown in Table 5. The modified H chain mutation region was called 2CAVHCOL1. The modified L chain variant region was called 2CAVLCOL1.

The resulting modified region gene was then purified by gel electrophoresis. To remove excess oligo and other incomplete DNA fragments that were not ligated, the ligated product was hanged on agarose gel at 1% low solubility at 100 V DC for 2 hours. Critical bands with full length DNA products were cut and placed in sterile 1.5 ml centrifuge tubes. To release the DNA from the agarose, the gel pieces were cut with f3-agarase I at 40 ° C. DNA was harvested by charging with 0.3 M NaOAc and isopropanol at −20 ° C. for 1 hour and then centrifuged at 12,000 rpm for 15 minutes. Purified DNA pellet was resuspended in 50 μl of TE buffer (pH 8.0). The processed mutation region gene was then amplified by PCR. In particular, 100 ng of the engineered mutagenesis gene was mixed with 25 mM dNTPs, 200 ng primer, 5 U high purity thermostable Pfu DNA polymerase in buffer. The resulting PCR product was analyzed on 1% agarose gel.

Each purified DNA corresponding to the processing variant region gene was subsequently inserted into the pUC 19 bacterial vector. pUC19 is a 2686 base pair and a large number of E. coli plasmid vectors with 54 base pair polylinks on the lacZ and Amp selection marks. To make a vector for insertion of the engineered mutation region gene, 10 μg of pUC19 was linearized with Hinc II (50U) at 37 ° C. for 3 hours to produce a vector with blunt terminal 5′GTC. To prevent self-ligation, linear vector DNA was dephosphorylated with 25 U of bovine visceral alkaline phosphatase (CIP) at 37 ° C. for 1 hour. To insert the engineered variant region gene into the pUC19 vector, approximately 0.5 μg of dephosphorylated linear vector DNA was mixed with 3 μg of phosphorylated variant region gene in the presence of T4 DNA ligase (1000U) and 16 for 12 hours. Incubated at ℃.

Bacterial vectors carrying the engineered variant region genes were then used to transform bacterial cells. In particular, 50 μl of freshly produced strong DH-5α cells were mixed with 1 μg of pUC19 carrying the engineered mutagenesis gene and transferred to an electroporation cuvette (0.2 cm gap; Bio-Rad). Each cuvette was pulsed at 2.5 kV / 200 ohm / 25 Hz using an electroporation device ((Bio-Rad Gene Pulser). Then, 1 ml of SOC medium was added to each cuvette, and the cells were placed in a centrifuge tube. The cells were recovered for 1 hour at 37 ° C. A portion of each transformed cell was removed, diluted 1: 100, and then 100 μl plated onto an LB plate containing ampicillin (Amp 40 μg / ml). Due to the presence of the mark it was incubated overnight at 37 ° C. Only transformants carrying the pUC19 vector grew on LB / Amp plates.

Monotransformer colonies were selected and continuously shaken at 37 ° C. with overnight incubation in 3 ml LB / amp sterile glass tubes. Plasmid DNA was isolated using an Easy Prep column (Pharmacia Biotech) and suspended in 100 μl of TE buffer (pH7.5). To confirm the presence of gene insertion on pUC19, 25 μl of plasmid DNA from each colony was digested with restriction enzymes for 1 hour at 37 ° C. and analyzed on a 1% agarose gel. In this way, the plasmid DNA gene insert is resistant to enzymatic cleavage due to the loss of restriction sites (5 '.. GTCGAC..3') and becomes closed circular (CC) DNA, but the plasmid without insertion is cleaved and gelled. Becomes a linear (L) two-stranded DNA fragment.

DNA sequencing was performed on an automated ABI 377 DNA sequencing device for M13 / pUC reversed-phase primers (5'AACAGCTATGACCATG3 ') to identify the exact gene sequence of the engineered variant region gene and to eliminate the possibility of unwanted mutations made during construction. ) And PCR gene products are also carried out using 5 'terminal 20 base pair primer (5'GAATT CATGGCTTG GGTGTG 3').

Table 5

Construction of genes encoding modified antibodies with CDRs from Mab 31.1

2CAVHCOLI Oligo1VHC1 Oligo2VHC2 Oligo3VHC3 Oligo4VHC4 Oligo5VHC5 Oligovhc Oligo7VHC7 Oligo8VHC8 Oligo9VHC9 Oligo10VHC10 2CAVLCOLI VLC1 VLC2 VLC3 VLC4 VLC5 VLC VLC7 VLC8 VLC9 VLC10

6.3. Insertion of engineered mutation region gene into mammalian expression vector

Complete antibody L chains possess both mutation and constant regions. The full antibody H chain has a mutation region, a constant region, and a hinge region. The modified variant region gene 2CAVHCOL1 or 2CAVLCOL1 was inserted into a vector having an appropriate constant region. The engineered mutagenesis gene lacking cysteine residues in the L chain was inserted into the pMRRO10.1 vector (FIG. 6A). The pMRRO10.1 vector had a human Kappa L chain constant region. The insertion of the engineered L chain mutation region into this vector produced the complete L chain sequence. Alternatively, the engineered variant region gene lacking the cysteine residue on the H chain was inserted into the pGAMMA1 vector (FIG. 6B). The pGAMMA1 vector possessed human and IgG1 constant region, hinge region sequences. Insertion of the processed H chain variant region gene into this vector resulted in the complete H chain sequence.

To process mammalian vectors consisting of H chain and L chain genes, the complete L chain sequence and the complete H chain sequence were inserted into the mammalian expression vector pNEPuDGV (FIG. 6C) (Bebbington, CR, 1991, In METHODS: A Companion). to Methods in Enzymology, vol. 2, pp. 136-145). The resulting vector encodes both the H and L chains of the modified antibody.

6.4. Expression of Artificially Modified Antibodies in Mammalian Cells

To test the production of the combined antibodies, mammalian expression vectors were transfected with COS cells. COS cells (transformed with the African Green Monkey Renal Cell Line, CV-1, origin-deficient SV40 virus) are used for short term transient expression of artificial antibodies because they have a large number of copies of circular plasmids bearing replication of SV40 origin. Because it can be replicated. Antibody expression vectors were transferred to C0S7 cells (American Type Culture Collection). The transferred vectors were grown in Dulbecco's modified Eagle's medium and transfected with expression vectors using calcium precipitation (Sullivan et al., FEBS Lett. 285: 120-123, 1991). Transfected cells were cultured 72 hours after supernatant harvest. Supernatants of transfected COS were analyzed by ELISA method for combined IgG. ELISA includes capture of samples and references to 96-well plates, which are coated with anti-human IgG Fc. Bound Combination IgG was detected with anti-human Kappa chains bound to horse radish epoxidase (HRP) and tetramethylbenzidine substrate (TMB). The color change was proportional to the amount of combination antibody present on the sample.

6.5. Modified antibodies that immunospecifically bind to human colon cancer (species) cells and antigens produced by these cells

Modified antibodies were expressed and isolated as indicated in section 6.4. Binding capacity and specificity were then analyzed using LS-174T cell WiDR cells (human colon cancer cell line) and antigens derived from these cells.

To check MA31.1 binding specificity and binding capacity, dot blot analysis was performed (FIG. 11). Membrane compositions obtained on LS-174T cells were applied to nitrocellulose membranes using a Bio-Blot device (Bio-Rad). Wells blocked non-specific binding with steaming milk. Physiologically miniaturized antibodies derived from Mab31.1 were cultured in all wells. Unlabeled antibodies at a concentration of 0.003 to 20 nM were then applied to the nitrocellulose membrane and incubated. Unbound antibody was washed away from the membrane and a second antibody, alkaline phosphatase labeled anti-human IgG, was added. Finally, addition of an alkaline phosphatase substrate results in a dark purple precipitate, suggesting the presence of bound labeling antibody. 11 provides the results of the dot blot analysis. The figures demonstrated that the labeled antibody binds to LS-174T cells. In addition, unlabeled antibodies compete with physiologically miniaturized antibodies, suggesting the specificity of Mab31.1-derived antibodies for tumor cell antigens.

6.6. Anti-genetic reactions

The binding effect of the modified antibodies on LS-174T cells was examined in a competitive binding assay. LS-174T cells are human colon cancer (species) cells that express antibodies recognized by the Mab31.1 antibody. Peptides with one sequence of the CDRs of the Mab31.1 antibody were made. These peptides, modified antibodies, and control antibodies derived from Mab31.1 were administered to mice to produce antiserum and antibodies against the CDRs of Mab31.1. Blood samples from mice were drawn 2 weeks and 42 weeks after injection. Antisera of immunized mice were used for binding competition assay (FIGS. 12A, B).

Antisera and physiologically miniaturized antibodies were analyzed for their ability to bind LS-174T cells. As shown in FIGS. 12A and B, antiserum generated against CDR3, CDR4 peptides was in fierce competition with control antibodies (antibodies derived from Mab31.1) that bind to LS-174T cells. Antisera generated against CDR1, CDR2 also competed significantly with control antibodies that bind to LS-174T cells (FIG. 12B). In addition, antiserum of mice injected with 2CAVHCOL1 and 2CAVLCOL1 antibodies (ie, antibodies in which cysteine was converted to alanine in the mutant region) was more effective than physiologically derived from Mab31.1 than antiserum of mice injected with Mab31.1-induced antibody. Competed with miniaturized antibodies. In these results, administration of an antibody whose cysteine is changed to alanine in the mutation region can induce anti-genetic antibodies that recognize colon cancer (species) cell antigens more effectively than antibodies having chain-to-disulfide bonds in the mutation region. It can be seen that.

Table 6

Biotin-labeled Peptides Derived from Mab 31.1 CDR Sequences

Peptide ID sequence

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

COL311H2 Biotin-N-Ala-Gly-Trp-Ile-Asn-Thr-Tyr-Thr-Gly-Glu-Pro-Thr-Tyr-

Ala-Asp-Asp-Phe-Lys-Gly

COL311 H3 Biotin-N-Ala-Arg-Ala-Tyr-Tyr-Gly-Lys-Tyr-Phe-Asp-Tyr

Example 7: Production of Artificially Modified Antibodies Containing HMFG-1 Sequences

Anti-genetic antibodies were constructed that immunospecifically bind to HMFG-1 antibodies. HMFG-1 is an antibody known to bind to polymorphic epithelial muchins (PEM) (Stewart et al., 1990, J Clin Oncol 8: 1941-50; Kosmas et al., 1994, Cancer 73: 3000-3010). The antigenic determinant of PEM having the ProAspThrArgPro sequence was inserted into the modified chain region by the method of the present invention. This short sequence is the highly immunogenic region of human polymorphic epithelial mucins (Gendler et al., 1988, J. Biol. Chem. 263: 12820-12823). Residues 27A-27E (SerLeuLeuTyrSer) of HMFG-1 (Table 6) were substituted with ProAspThrArgPro using the oligonucleotide method identified in section 6 (FIG. 10). Anti-genetic artificial HMFG-1 antibodies that immunospecifically bind to HMFG-1 were made using known sequences of the variant regions of the L and H chains of HMFG-1. Oligos were added in the following 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 the sequence comparison between HMFG-1 and various consensus CDR sequences. Information on HMFG-1 and related monoclonal antibodies is presented in WO 09/05142 (Imperial Cancer Research Technology, Ltd), and immunized HMFG-1 is presented in WO 92/04380 (Unilever).

Polymerase sequencing (PCR) was used to amplify the combined sequence (FIG. 13). The processing variant region constructed to retain the nucleotide sequence encoded by HMFG-1 is shown in FIG. The engineered variant region gene was inserted into a vector suitable for antibody production, such as the pNEFuDGV vector (section 6). Other methods of constructing engineered genes can be used, such as 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, but are not limited to these.

TABLE 7

The present invention is not to be limited in scope by the specific embodiments presented in this article. Indeed, various modifications of the invention, including those set forth herein, will be apparent to those skilled in the art upon review of the foregoing specification and accompanying drawings. Such modifications fall within the scope of the following claims.

The various resources cited in this article are hereby incorporated by reference in their entirety.

Claims (98)

In a vaccine composition comprising a sufficient amount of a first immunoglobulin molecule and a pharmaceutically acceptable carrier for inducing an anti-genetic response, the first immunoglobulin molecule comprises a variant region, wherein one or multiple amino acids are present in the variant region. Is identical to the second immunoglobulin molecule except that it is substituted, the second immunoglobulin molecule may immunospecifically bind to the antigen, and the one or plural amino acid substitutions do not have an SH group at one or plural positions. Vaccine composition, characterized in that the substitution of one or multiple amino acid residues, wherein the position corresponds to one or multiple cysteine residues that form a disulfide bond to the immunoglobulin molecule. The vaccine composition according to claim 1, wherein the antigen is a tumor antigen. The vaccine composition according to claim 2, wherein the antigen is a cancer antigen. 4. The vaccine composition of claim 3 wherein the antigen is a polymorphic endothelial mucin antigen. 4. The vaccine composition of claim 3 wherein the antigen is a human colon cancer-related protein antigen. 4. The vaccine composition of claim 3 wherein the antigen is human colon cancer-related carbohydrate antigen. The method of claim 1, wherein the mutation region is L (light) chain mutation region, wherein the amino acid residue having no SH group is located at a position corresponding to the 23 or 88 position of the L chain mutation region of the second immunoglobulin molecule Vaccine composition. The method of claim 1, wherein the mutation region is H (heavy) chain mutation region, wherein the amino acid residue having no SH group is located at a position corresponding to position 22 or 92 of the H chain mutation region of the second immunoglobulin molecule Vaccine composition. The vaccine composition according to claim 1, 7, or 8, wherein the amino acid residue having no SH group is alanine. The method of claim 1, wherein the second immunoglobulin molecule is Mab 31.1, Mab 33.28 or Mab HMFG-1, wherein one or plural amino acid substitutions include substitution with alanine at positions 23 or 88 of the L-chain variant region. Vaccine composition. The method of claim 1, wherein the second immunoglobulin molecule is Mab 31.1, Mab 33.28 or Mab HMFG-1, wherein one or more amino acid substitutions include a substitution with alanine at positions 22 or 92 of the H-chain variant region. Vaccine composition. 4. The vaccine composition of claim 3 wherein the antigen is a human milk fat sphere antigen. The vaccine composition according to claim 3, wherein the antigen is a cancer antigen of breast, ovary, uterus, prostate, bladder, lung, skin, colon, pancreas, gastrointestinal tract, B cell or T cell. The antigen according to claim 3, wherein the antigen is KS 1/4 pan-carcinoma antigen, ovarian carcinoma antigen, prostate phosphate, prostate specific antigen, melanoma-associated antigen P97, melanoma antigen GP75, high molecular weight melanoma antigen, nodule Specific membrane antigens, fetal carcinoma antigens, polymorphic endothelial intact antigens, human milk fat specific antigens, colorectal tumor-associated antigens TAG-72, CO17-1A, GICA 19-9, CTA-1, LEA, Burkitt Lymphoma antigen-38.13, CD19, human B-lymphoma antigen-CD20, CD33, ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside GM3, tumor-specific graft cell-surface antigen, neotumor Antigen-alpha-fetoprotein L6, human lung carcinoma antigen L20, human leukemia T cell antigen-GP37, glycoprotein, sphingolipids, EGFR, HER2 antigen, polymorphic endothelial, malignant human lymphocyte antigen-APO-1, antigen M18, M39, SSEA-1, VEP8, VEP9, Myl, VIM-D5, D56-22, TRA-1-85, C14, F3, AH6, Y hapten, Le ^y , TL5, FC10.2, gastric adenocarcinoma antigen, C0 -514, NS-10, CO-43, MH2, 19.9 found in colon cancer, gastric cancer, T5A7, R24, 4.2, GP3, D1.1, OFA-1, GM2, OFA-2, G _P2 , M1: 22 : 25: 8, SSEA-3, SSEA-4, T-cell receptor inducing peptides. The method of claim 1, wherein the antigen is an antigen of an infectious disease pathogen. The antigen according to claim 15, wherein the antigen is influenza virus hemagglutinin, human respiratory syncytial virus G glycoprotein, deep protein of dengue virus, parent protein of dengue virus, measles virus hemagglutinin, monocytovirus virus type 2 glycoprotein gB, poliovirus I VP1, envelope glycoprotein of HIV I, hepatitis B surface antigen, diphtheria toxin, Streptococcus 24M epitope, Gonococcal pilin, Pseudorabies virus g50, Pseudorabies virus glycoprotein H, Pseudorabies virus glycoprotein E, spreadable gastroenteritis glycoprotein 195, spreadable gastroenteritis maternal protein, porcine rotavirus glycoprotein 38, porcine parvovirus capsid protein, sulfuryl hydrodicente Sera (Serpulina hydodysenteriae) protective antigen, bovine viral diarrhea glycoprotein 55, Newcastle disease virus hemagglutinin Uraminidase, swine cold hemagglutinin, swine cold neuraminidase, infectious bovine nasal bronchitis virus glycoprotein E, infectious laryngitis virus glycoprotein G or glycoprotein I, glycoprotein of La Crosse virus , Fetal diarrhea virus, hepatitis B virus deep 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 A Type / Kentucky 81 Neuuraminidase, Equine Herpesvirus Type 1 Glycoprotein B, Equine Herpesvirus Type 1 Glycoprotein D, Bovine Respiratory Synthetic Virus Adhesion Protein, Bovine Respiratory Synthetic Virus Fusion Protein, Bovine Respiratory Synthetic Virus Nucleotide Capsid Protein, Bovine Parainfluenza Virus Type 3 Fusion Protein, Bovine Parainfluenza Bars A vaccine composition characterized in that it is selected from the virus type 3 hemagglutinin neuraminidase, bovine viral diarrhea virus glycoprotein 48, bovine diarrhea virus glycoprotein 53. The vaccine composition of claim 1, wherein the antigen is a cell receptor for an infectious disease pathogen. 18. The method of claim 17, wherein the cell receptor is LPV receptor, adenylate silase, BDV surface glycoprotein, N-acetyl-9-O-acetylneuraminic acid receptor, CD4 +, highly sulfated heparin sulfate, p65, Gal alpha 1-4-Gal-containing isotope, CD16b, integrin VIM-2 receptor, EV receptor, CD14, glycoconjugate receptor, alpha / beta T-cell receptor, corrosion-accelerating factor receptor, extracellular envelope glycoprotein receptor, immunoglobulin Fc Receptor Paxvirus M-77, GALV Receptor, CD14 Receptor, Lewis (b) Blood Group Antigen Receptor, T-Cell Receptor, Heparin Sulfate Glycoaminoglycan Receptor, Fibroblast Growth Factor Receptor, CD11a, CD2, G-protein Binding Receptor, CD4, heparin sulfate proteoglycan, Annexin II, CD13 (aminopeptidase N), human aminopeptidase N receptor, hemagglutinin receptor, CR3 receptor, protein kinase receptor, galactose N-acetylgalactosamine -Injury Sex lectin receptor, chemokine receptor, annexin I, actA protein, CD46 receptor, meningococcal toxicity related opa receptor, CD46 receptor, fetal carcinoma antigen family receptor, fetal carcinoma antigen family Bgla receptor, gamma interferon receptor, glycoprotein gp70, rmc-1 receptor, human integrin receptor alpha v beta 3, heparin sulfate floateoglycan receptor, CD66 receptor, integrin receptor, membrane cofactor protein, CD46, GM1, GM2, GM3, CD3, ceramide, hemagglutinin -Neuuraminidase protein, erythrocyte P antigen receptor, CD36 receptor, glycophorin A receptor, interferon gamma receptor, KDEL receptor, mucosal homing alpha4beta7 receptor, endothelial growth factor receptor, alpha5beta1 integrin protein, non-sugar J774, CXCR1-4 Receptor, CCR1-5 Receptor, CXCR3 Receptor, CCR5 Receptor, He46 Saturated Glycoprotein, TNFR P55 Receptor, TNFp75 Receptor, Water Soluble Interleukin-1 A vaccine composition characterized in that the selection at the other receptors. 18. The vaccine composition according to claim 15 or 17, wherein the infectious disease pathogen is a bacterium. 20. The bacterium of claim 19, wherein the bacterium is Mycobateria rickettsia, Mycoplasma, Neisseria spp., Legionella, Shigella spp., Vibrio cholera. cholerae, Streptococci, Cornebacteria diphtheriae, Clostridium tetani, Bordetella pertussis, Haemophilus spp., Chlamydia species Chlamydia spp.,), Escherichia coli, or a vaccine composition characterized by causing syphilis or Lyme disease. 18. The vaccine composition according to claim 15 or 17, wherein the infectious disease pathogen is a virus. The method of claim 21, wherein the virus is hepatitis A, hepatitis B, hepatitis C, influenza, chickenpox, adenovirus, herpes simplex virus type I, herpes simplex type II, aerobic, nasal cold virus, echovirus, rotavirus, Respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, hantan virus, coxsackie virus, mumps virus, measles virus, rubella virus, poliovirus, human immunodeficiency virus I Type, human immunodeficiency virus type II, picornavirus, enterovirus, calciviridia, norwalk virus group, togavirus, alphavirus, flavivirus, coronavirus, laviz virus, marburg virus, ebolavirus , Parainfluenza virus, orthomyxovirus, field virus, are Virus, leovirus, rotavirus, orbivirus, human T cell leukemia virus type I, human T cell leukemia virus type II, apes immunodeficiency virus, lentivirus, polooma virus, parvovirus, Epstein-Barr virus, human Vaccine composition, characterized in that selected from herpesvirus-6, monkey herpesvirus 1, Paxvirus. 18. The vaccine composition according to claim 15 or 17, wherein the infectious disease pathogen is a parasite. 24. The vaccine composition according to claim 23, wherein the parasite is selected from malaria, ymeria, resimania, coccidioea, trimansomoma, and fungus. The vaccine composition of claim 1, wherein the first immunoglobulin is selected from IgG, IgE, IgM, IgD, IgA. In a vaccine composition comprising a sufficient amount of a first immunoglobulin molecular fragment and a pharmaceutically acceptable carrier for inducing an anti-genetic response, the first immunoglobulin molecular fragment comprises a variant region, one or more of which is present in the variant region. Same as the corresponding fragment of the second immunoglobulin molecule, except that the plural amino acid is substituted, the fragment of the second immunoglobulin molecule may immunospecifically bind to the antigen, and the one or plural amino acid substitution is one or Vaccine composition, characterized in that the substitution of one or a plurality of amino acid residues having no SH group in a plurality of positions, wherein the position corresponds to one or multiple cysteine residues that form a disulfide bond to the immunoglobulin molecule. 27. The vaccine composition of claim 26 wherein the fragment is a single chain immunoglobulin. 27. The vaccine composition of claim 26 wherein the fragment is a Fab fragment, a (Fab ') 2 fragment, an H chain dimer, an L chain dimer or an Fv fragment. 27. The vaccine composition of claim 26 wherein the fragment also comprises a region. 27. The vaccine composition according to claim 26, wherein the mutant region is obtained from mouse immunoglobulin and the predetermined region is obtained from human immunoglobulin. The vaccine composition according to claim 1, wherein the variant region has a framework region from a human antibody and a complementary determining region (CDR) from a mouse immunoglobulin. The method of claim 1, wherein the first immunoglobulin molecule is coupled to the amino acid sequence of the protein through covalent bonds, the protein is Il-2, Il-4, Il-5, Il-, Il-7, Il-10, Vaccine composition, characterized in that it is selected from interferon or MHC-induced peptide, G-CSF, TNF, porin, NK cell antigen or cellular endocytosis receptor. The fragment of claim 26, wherein the fragment of the first immunoglobulin molecule binds to an amino acid sequence of the protein via a covalent bond, wherein the protein is Il-2, Il-4, Il-5, Il-5, Il-7, Il-7, Il-. 10, Vaccine composition, characterized in that it is selected from interferon or MHC-induced peptide, G-CSF, TNF, porin, NK cell antigen or cellular endocytosis receptor. A method of inducing an anti-genetic response in a subject, the method comprising administering to the subject a sufficient amount of a first immunoglobulin molecule to induce an anti-genetic response, wherein the first immunoglobulin molecule is mutated. A region, the same as a second immunoglobulin molecule, except that one or a plurality of amino acids are substituted in the variant region, and the second immunoglobulin molecule may immunospecifically bind to an antigen, wherein the one or The plural amino acid substitution is a substitution of one or plural amino acid residues having no SH group at one or plural positions, wherein the position corresponds to one or plural cysteine residues forming a disulfide bond in the immunoglobulin molecule. 35. The method of claim 34, wherein said method comprises separating an antibody from an individual, said antigen recognizing the genotype of a second immunoglobulin molecule, and administering said antigen to said second individual. The method of claim 34, wherein the antigen is a tumor antigen. The method of claim 34, wherein the antigen is a cancer antigen. The method of claim 36, wherein the antigen is a polymorphic endothelial intact antigen. 37. The method of claim 36, wherein the antigen is human colon cancer (species) -associated protein antigen. 37. The method of claim 36, wherein the antigen is human colon cancer (species) -related carbohydrate antigen. The method of claim 35, wherein the mutation region is L (light) chain mutation region, wherein the amino acid residue having no SH group is located at a position corresponding to the 23 or 88 position of the L chain mutation region of the second immunoglobulin molecule How to. The method of claim 35, wherein the mutation region is H (heavy) chain mutation region, wherein the amino acid residue having no SH group is located at a position corresponding to position 22 or 92 of the H chain mutation region of the second immunoglobulin molecule How to. 35. The method of claim 34, wherein the amino acid residue having no SH group is alanine. 35. The method of claim 34, wherein the second immunoglobulin molecule is Mab 31.1, Mab 33.28 or Mab HMFG-1, and the amino acid substitutions comprise substitution of alanine at positions 23 or 88 of the L-chain variant region. 35. The method of claim 34, wherein the second immunoglobulin molecule is Mab 31.1, Mab 33.28 or Mab HMFG-1, and wherein the amino acid substitutions include substitution with alanine at positions 22 or 92 of the H chain variant region. The method of claim 34, wherein the antigen is a human milk fat sphere antigen. 35. The method of claim 34, wherein the antigen is an antigen for cancer of the breast, ovary, uterus, prostate, bladder, lung, skin, colon, pancreas, gastrointestinal tract, B cell or T cell. The method of claim 36, wherein the antigen is KS 1/4 pan-carcinoma antigen, ovarian carcinoma antigen, prostate phosphate, prostate specific antigen, melanoma-associated antigen P97, melanoma antigen GP75, high molecular weight melanoma antigen, nodule Specific membrane antigens, fetal carcinoma antigens, polymorphic endothelial intact antigens, human milk fat specific antigens, colorectal tumor-associated antigens TAG-72, CO17-1A, GICA 19-9, CTA-1, LEA, Burkitt Lymphoma antigen-38.13, CD19, human B-lymphoma antigen-CD20, CD33, ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside GM3, tumor-specific graft cell-surface antigen, neotumor Antigen-alpha-pepprotein L6, human lung carcinoma antigen L20, human leukemia T cell antigen-GP37, neoglycoprotein, sphingolipids, EGFR, HER2 antigen, polymorphic endothelial, malignant human lymphocyte antigen-APO-1, antigen M18 , M39, SSEA-1, VEP8, VEP9, Myl, VIM-D5, D56-22, TRA-1-85, C14, F3, AH6, Y hapten, Le ^y , TL5, FC10.2, gastric adenocarcinoma antigens, C0-514, NS-10, CO-43, MH2, 19.9 found in colon cancer, gastric cancer, T5A7, R24, 4.2, GP3, D1.1, OFA-1, GM2, OFA-2, G _P2 , M1: 22: 25: 8, SSEA-3, SSEA-4, T-cell receptor inducing peptide. 35. The method of claim 34, wherein the antigen is an antigen of an infectious disease pathogen. 50. The method of claim 49, wherein the antigen is influenza virus hemagglutinin, human respiratory syncytial virus G glycoprotein, deep protein of dengue virus, parent protein of dengue virus, measles virus hemagglutinin, monocytovirus virus type 2 glycoprotein gB, poliovirus I VP1, envelope glycoprotein of HIV I, hepatitis B surface antigen, diphtheria toxin, Streptococcus 24M epitope, gonococcal pilin, Pseudorabies virus g50 , Pseudorabies virus glycoprotein H, Pseudorabies virus glycoprotein E, spreadable gastroenteritis parent protein, porcine rotavirus glycoprotein 38, porcine parvovirus capsid protein, Sulfurina hydodysenteriae protective antigen, Bovine virus diarrhea glycoprotein, swine cold neuraminidase, infectious bovine nasal bronchitis virus glycoprotein E, persimmon Laryngitis Virus Glycoprotein G or Glycoprotein I, Glycoprotein of La Crosse Virus, Fetal Diarrhea Virus, Hepatitis B Deep Protein, Hepatitis B Surface Antigen, Equine Influenza Virus Type A / Alaska 91 New Uramidase, Equine Influenza Virus Type A / Miami 63 New Uramidase, Equine Influenza Virus Type A / Kentucky 81 New Uramidase, Equine Herpesvirus Type 1 Glycoprotein B, Equine Herpes Virus Type 1 Glycoprotein D, Bovine Respiratory Tract Concomitant Virus Fusion Protein, Bovine Respiratory Combination Virus Nucleocapsid Protein, Bovine Praffininfluenza Virus Type 3 Fusion Protein, Bovine Paraffininfluenza Virus Type 3 Hemagglutinin Nuuraminidase, Bovine Viral Diarrhea Virus Glycoprotein 48, And bovine diarrhea glycoprotein 53. 35. The method of claim 34, wherein the antigen is a cell receptor for an infectious disease pathogen. 52. The cell receptor of claim 51, wherein the cell receptor is an LPV receptor, adenylate silase, BDV surface glycoprotein, N-acetyl-9-O-acetylneuraminic acid receptor, CD4 +, highly sulfated heparin sulfate, p65, Gal alpha. 1-4-Gal-containing isotope, CD16b, integrin VLA-2 receptor, EV receptor, CD14, glycoconjugate receptor, alpha / beta T-cell receptor, corrosion-accelerating factor receptor, extracellular envelope glycoprotein receptor, immunoglobulin Fc receptor, faxvirus M-77, GALV receptor, CD14 receptor, Lewis (b) blood group antigen receptor, T-cell receptor, heparin sulfate glycoaminoglycan receptor, fibroblast growth factor receptor, CD11a, CD2, G-protein Binding receptor, CD4, heparin sulfate proteoglycan, Annexin II, CD13 (aminopeptidase N), human aminopeptidase N receptor, hemagglutinin receptor, CR3 receptor, protein kinase receptor, galactose N-acetylgalacto Samine-I Sex lectin receptor, chemokine receptor, annexin I, actA protein, CD46 receptor, meningococcal toxicity related opa receptor, CD46 receptor, fetal carcinoma antigen family receptor, fetal carcinoma antigen family Bgla receptor, gamma interferon receptor, glycoprotein gp70, rmc-1 receptor, human integrin receptor alpha v beta 3, heparin sulfate floateoglycan receptor, CD66 receptor, integrin receptor, membrane cofactor protein, CD46, GM1, GM2, GM3, CD3, ceramide, hemagglutinin -Neuuraminidase protein, erythrocyte P antigen receptor, CD36 receptor, glycophorin A receptor, interferon gamma receptor, KDEL receptor, mucosal homing alpha4beta7 receptor, endothelial growth factor receptor, alpha5beta1 integrin protein, non-sugar J774, CXCR1-4 Receptor, CCR1-5 Receptor, CXCR3 Receptor, CCR5 Receptor, He46 Saturated Glycoprotein, TNFR P55 Receptor, TNFp75 Receptor, Water Soluble Interleukin-1 Characterized in that it is selected from beta receptors. 52. The method of claim 49 or 51, wherein the infectious disease agent is a bacterium. 54. The bacterium of claim 53, wherein the bacterium is Mycobateria rickettsia, Mycoplasma, Neisseria spp., Legionella, Shigella spp., Vibrio cholera. cholerae, Streptococci, Cornebacteria diphtheriae, Clostridium tetani, Bordetella pertussis, Haemophilus spp., Chlamydia species Chlamydia spp.,), Escherichia coli, or characterized by causing syphilis or Lyme disease. 52. The method of claim 49 or 51, wherein the infectious disease agent is a virus. 55. The method of claim 53, wherein the virus is hepatitis A, hepatitis B, hepatitis C, influenza, chickenpox, adenovirus, herpes simplex virus type I, herpes simplex type II, aerobic, nasal cold virus, echovirus, rotavirus, Respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, hantan virus, coxsackie virus, mumps virus, measles virus, rubella virus, poliovirus, human immunodeficiency virus I Type, human immunodeficiency virus type II, picornavirus, enterovirus, calciviridia, norwalk virus group, togavirus, alphavirus, flavivirus, coronavirus, laviz virus, marburg virus, ebolavirus , Parainfluenza virus, orthomyxovirus, field virus, arena Virus, leovirus, rotavirus, orbivirus, human T cell leukemia virus type I, human T cell leukemia virus type II, apes immunodeficiency virus, lentivirus, polooma virus, parvovirus, Epstein-Barr virus, human Herpesvirus-6, monkey herpesvirus 1, faxvirus. 52. The method of claim 49 or 51, wherein the infectious disease pathogen is a parasite. 58. The method of claim 57, wherein the parasite is selected from malaria, ymeria, resimania, coccidioa, trimanosoma, mold. The method of claim 34, wherein the first immunoglobulin is selected from IgG, IgE, IgM, IgD, IgA. In a method of inducing an anti-genetic response in a subject, the method comprises administering to the individual a sufficient amount of a first immunoglobulin molecular fragment to induce an anti-genetic response, the fragment comprising a variant region. And a fragment of the second immunoglobulin molecule, except that one or a plurality of amino acids are substituted in the mutant region, and the fragment of the second immunoglobulin molecule may immunospecifically bind to an antigen. Or plural amino acid substitutions are substitutions of one or plural amino acid residues having no SH group at one or plural positions, wherein the positions correspond to one or plural cysteine residues which form disulfide bonds to the immunoglobulin molecule. 61. The method of claim 60, wherein said method comprises separating an antibody from an individual, said antigen recognizing the genotype of a second immunoglobulin molecule, and administering said antigen to said second individual. 61. The method of claim 60, wherein the antigen is a tumor antigen. 61. The method of claim 60, wherein the antigen is a cancer antigen. 61. The method of claim 60, wherein the antigen is a polymorphic endothelial intact antigen. 63. The method of claim 62, wherein the antigen is human colon cancer (species) -associated protein antigen. 63. The method of claim 62, wherein the antigen is human colon cancer (species) -related carbohydrate antigen. 61. The method of claim 60, wherein the mutation region is L (light) chain mutation region, amino acid residues having no SH group is located at a position corresponding to position 23 or 88 of the L chain mutation region of the second immunoglobulin molecule How to. 61. The method of claim 60, wherein the mutation region is H (heavy) chain mutation region, amino acid residues having no SH group is located at a position corresponding to position 22 or 92 of the H chain mutation region of the second immunoglobulin molecule How to. 69. The method of claim 60, 67 or 68, wherein the amino acid residue having no SH group is alanine. 64. The method of claim 63, wherein the antigen is a human milk fat sphere antigen. 64. The method of claim 63, wherein the antigen is an antigen for cancer of the breast, ovary, uterus, prostate, bladder, lung, skin, colon, pancreas, gastrointestinal tract, B cell or T cell. 61. The method of claim 60, wherein the antigen is an antigen of an infectious disease pathogen. 73. The antigenic antibody of claim 72, wherein the antigen is influenzavirus hemagglutinin, human respiratory syncytial virus G glycoprotein, deep protein of dengue virus, parent protein of dengue virus, measles virus hemagglutinin, monocytic virus type 2 glycoprotein gB, poliovirus I VP1, envelope glycoprotein of HIV I, hepatitis B surface antigen, diphtheria toxin, Streptococcus 24M epitope, Gonococcal pilin, Pseudorabies virus g50, Pseudorabies virus glycoprotein H, Pseudorabies virus glycoprotein E, spreadable gastroenteritis glycoprotein 195, spreadable gastroenteritis maternal protein, porcine rotavirus glycoprotein 38, porcine parvovirus capsid protein, sulfuryl hydrodicente Sera (Serpulina hydodysenteriae) protective antigen, bovine viral diarrhea glycoprotein 55, Newcastle disease virus hemagglutinin Uraminidase, swine cold hemagglutinin, swine cold neuraminidase, infectious bovine nasal bronchitis virus glycoprotein E, infectious laryngitis virus glycoprotein G or glycoprotein I, glycoprotein of La Crosse virus , Fetal diarrhea virus, hepatitis B virus deep 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 A Type / Kentucky 81 Neuuraminidase, Equine Herpesvirus Type 1 Glycoprotein B, Equine Herpesvirus Type 1 Glycoprotein D, Bovine Respiratory Synthetic Virus Adhesion Protein, Bovine Respiratory Synthetic Virus Fusion Protein, Bovine Respiratory Synthetic Virus Nucleotide Capsid Protein, Bovine Parainfluenza Virus Type 3 Fusion Protein, Bovine Parainfluenza Bars The method is characterized in that it is selected from the virus type 3 hemagglutinin neuraminidase, bovine viral diarrhea virus glycoprotein 48, bovine diarrhea virus glycoprotein 53. 61. The method of claim 60, wherein the antigen is a cell receptor for an infectious disease pathogen. 75. The cell receptor of claim 74, wherein the cell receptor is a LPV receptor, adenylate silase, BDV surface glycoprotein, N-acetyl-9-O-acetylneuraminic acid receptor, CD4 +, highly sulfated heparin sulfate, p65, Gal alpha. 1-4-Gal-containing isotope, CD16b, integrin VIM-2 receptor, EV receptor, CD14, glycoconjugate receptor, alpha / beta T-cell receptor, corrosion-accelerating factor receptor, extracellular envelope glycoprotein receptor, immunoglobulin Fc Receptor Paxvirus M-77, GALV Receptor, CD14 Receptor, Lewis (b) Blood Group Antigen Receptor, T-Cell Receptor, Heparin Sulfate Glycoaminoglycan Receptor, Fibroblast Growth Factor Receptor, CD11a, CD2, G-protein Binding Receptor, CD4, heparin sulfate proteoglycan, Annexin II, CD13 (aminopeptidase N), human aminopeptidase N receptor, hemagglutinin receptor, CR3 receptor, protein kinase receptor, galactose N-acetylgalactosamine -Injury Sex lectin receptor, chemokine receptor, annexin I, actA protein, CD46 receptor, Meningococcal toxicity related opa receptor, CD46 receptor, fetal carcinoma antigen family receptor, fetal carcinoma antigen family Bgla receptor, gamma interferon receptor , Glycoprotein gp70, rmc-1 receptor, human integrin receptor alpha v beta 3, heparin sulfate floatoglycan receptor, CD66 receptor, integrin receptor, membrane cofactor protein, CD46, GM1, GM2, GM3, CD3, ceramide, he Magglutinin-neuuraminidase protein, erythrocyte P antigen receptor, CD36 receptor, glycophorin A receptor, interferon gamma receptor, KDEL receptor, mucosal homing alpha4beta7 receptor, endothelial factor receptor, alpha5beta1 integrin protein, Non-glycosylated J774 Receptor, CXCR1-4 Receptor, CCR1-5 Receptor, CXCR3 Receptor, CCR5 Receptor, He46 Surface Glycoprotein, TNFR P55 Receptor, TNFp75 Receptor Wherein the emitter is selected from the rukin -1 beta receptors. 75. The vaccine composition of claim 72 or 74 wherein the infectious disease agent is a bacterium. 77. The bacterium of claim 76, wherein the bacterium is Mycobateria rickettsia, Mycoplasma, Neisseria spp., Legionella, Shigella spp., Vibrio cholera. cholerae, Streptococci, Cornebacteria diphtheriae, Clostridium tetani, Bordetella pertussis, Haemophilus spp., Chlamydia species Chlamydia spp.,), Escherichia coli, or induce syphilis or Lyme disease. 75. The method of claim 72 or 74, wherein the infectious disease agent is a virus. 79. The virus of claim 78, wherein the virus is hepatitis A, hepatitis B, hepatitis C, influenza, chickenpox, adenovirus, herpes simplex virus type I, herpes simplex II, aerobic, nasal cold virus, echovirus, rotavirus, Respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, hantan virus, coxsackie virus, mumps virus, measles virus, rubella virus, poliovirus, human immunodeficiency virus I Type, human immunodeficiency virus type II, picornavirus, enterovirus, calciviridia, norwalk virus group, togavirus, alphavirus, flavivirus, coronavirus, laviz virus, marburg virus, ebolavirus , Parainfluenza virus, orthomyxovirus, field virus, are Virus, leovirus, rotavirus, orbivirus, human T cell leukemia virus type I, human T cell leukemia virus type II, apes immunodeficiency virus, lentivirus, polooma virus, parvovirus, Epstein-Barr virus, human Herpesvirus-6, monkey herpesvirus 1, faxvirus. 75. The method of claim 72 or 74, wherein the infectious disease pathogen is a parasite. 81. The method of claim 80, wherein the parasite is selected from malaria, ymeria, resimania, coccidioa, trimanosoma, mold. 61. The method of claim 60, wherein the fragment is a single chain immunoglobulin. The method of claim 82, wherein the fragment is a Fab fragment, (Fab ′) 2 fragment, H chain dimer, L chain dimer, or Fv fragment. 61. The method of claim 60, wherein the fragment also includes a station. 85. The method of claim 84, wherein the variant region is obtained from mouse immunoglobulin and the predetermined region is obtained from human immunoglobulin. 61. The method of claim 60, wherein the variant region has a framework region from a human antibody and a complementary determining region (CDR) from a mouse immunoglobulin. A method of preventing and treating cancer in a subject in need thereof, wherein the method comprises a vaccine composition comprising a sufficient amount of a first immunoglobulin molecule and a pharmaceutically acceptable carrier to induce an anti-genetic response. The first immunoglobulin molecule comprises a mutation region, and is identical to the second immunoglobulin molecule except that one or a plurality of amino acids are substituted in the mutation region, and the second immunoglobulin molecule Is immunospecifically bound to an antigen of an infectious disease pathogen, wherein the one or plural amino acid substitutions are substitutions of one or plural amino acid residues having no SH group at one or plural positions, and the position is disulfide in the immunoglobulin molecule. Specially corresponding to one or multiple cysteine residues forming a bond How to be gong. A method of preventing and treating an infectious disease in an individual in need of preventing or treating an infectious disease, the method comprises a vaccine composition comprising a sufficient amount of a first immunoglobulin molecule and a pharmaceutically acceptable carrier to induce an anti-genetic response. The first immunoglobulin molecule comprises a variant region and is the same as the second immunoglobulin molecule, except that one or a plurality of amino acids are substituted in the variant region. The globulin molecule may immunospecifically bind to an antigen of an infectious disease pathogen, wherein the one or plural amino acid substitutions is a substitution of one or plural amino acid residues having no SH group at one or plural positions, and the position is the immunoglobulin molecule. One or multiple cysteine residues to form disulfide bonds in the Wherein the corresponding. A method of preventing and treating an infectious disease in an individual in need of preventing or treating an infectious disease, the method comprises a vaccine composition comprising a sufficient amount of a first immunoglobulin molecule and a pharmaceutically acceptable carrier to induce an anti-genetic response. The first immunoglobulin molecule comprises a variant region and is the same as the second immunoglobulin molecule, except that one or a plurality of amino acids are substituted in the variant region. Globulin molecules can immunospecifically bind to cellular receptors for infectious disease pathogens, wherein the one or plural amino acid substitutions are substitutions of one or plural amino acid residues having no SH group at one or plural positions, wherein the position is the immune One or more times to form disulfide bonds in the globulin molecule Characterized in that it corresponds to a stain residue. 89. The infectious disease of claim 88 or 89, wherein the infectious disease is syphilis, gonorrhea, AIDS, malaria, shigella, salmonella, hepatitis A, hepatitis C, Lyme arthritis, encephalitis, herpes, Gram-negative bacterial infection, Gram-positive bacterial infection, Pneumococcus infection. A method of making a first immunoglobulin molecule capable of sufficiently inducing an anti-genotype reaction, wherein the first immunoglobulin molecule comprises a mutation region, except that one or a plurality of amino acids are substituted in the mutation region. Same as the two immunoglobulin molecules, wherein the second immunoglobulin molecule is capable of immunospecifically binding to the antigen, wherein the one or plural amino acid substitutions are substitutions of one or plural amino acid residues having no SH group at one or plural positions Wherein the position corresponds to one or multiple cysteine residues that form a disulfide bond to the immunoglobulin molecule, (a) In a nucleic acid encoding a second immunoglobulin, a nucleotide encoding one or plural cysteine residues forming the disulfide bond is replaced with a nucleotide encoding one or plural amino acid residues having no SH group, Constructing nucleic acids encoding immunoglobulin molecules; (b) introducing the nucleic acid constructed in step (a) into the cell such that the first immunoglobulin molecule is expressed by the cell; (c) recovering the expressed first immunoglobulin molecule. 92. The method of claim 91, wherein the nucleotides are substituted by site mutagenesis. A method of making a first immunoglobulin molecule capable of sufficiently inducing an anti-genotype reaction, wherein the first immunoglobulin molecule comprises a mutation region, except that one or a plurality of amino acids are substituted in the mutation region. Same as the two immunoglobulin molecules, wherein the second immunoglobulin molecule is capable of immunospecifically binding to the antigen, wherein the one or plural amino acid substitutions are substitutions of one or plural amino acid residues having no SH group at one or plural positions Wherein the position corresponds to one or multiple cysteine residues that form a disulfide bond to the immunoglobulin molecule, (a) synthesizing a nucleic acid having an artificial gene encoding the first immunoglobulin molecule; (b) introducing the nucleic acid nucleic acid synthesized in step (a) into the cell such that the encoded first immunoglobulin molecule is expressed by the cell; (c) recovering the expressed first immunoglobulin molecule. 94. The method of claim 91 or 93, wherein the second immunoglobulin molecule is Mab 31.1 or Mab 33.28. 94. The method of claim 91 or 93, wherein the second immunoglobulin molecule is HMFG-1. In a vaccine composition comprising a sufficient amount of a first immunoglobulin molecule and a pharmaceutically acceptable carrier for inducing an anti-genetic response, the first immunoglobulin molecule comprises a variant region, wherein one or multiple amino acids are present in the variant region. Is identical to the second immunoglobulin molecule except that the second immunoglobulin molecule is immunospecifically bound to the antigen, wherein at least some of the one or multiple amino acid substitutions are in one or multiple positions Is a substitution of one or plural amino acid residues having no SH group in which the position corresponds to one or plural cysteine residues forming a disulfide bond in the second immunoglobulin molecule, wherein the disulfide bond in the second immunoglobulin molecule One or multiple positions corresponding to one or multiple cysteine residues that form In one amino acid substitution or not a plurality of amino acid residues substituted with no SH group is a vaccine composition, characterized in that failing to stabilize the change. A method of inducing an anti-genetic response in a subject, said method comprising administering to said individual a sufficient amount of a first immunoglobulin molecule to induce an anti-genetic response, wherein said first immunoglobulin molecule is mutated. A fragment of a second immunoglobulin molecule, wherein the second immunoglobulin molecule is immunospecifically bound to an antigen, wherein the fragment comprises a region, except that one or multiple amino acids are substituted for the variant region. At least one of the one or plural amino acid substitutions is a substitution of one or plural amino acid residues having no SH group at one or plural positions, wherein the position is at one or plural cysteine residues which form a disulfide bond to the second immunoglobulin molecule. In this case, one or forming a disulfide bond in the second immunoglobulin molecule Number in one or a plurality of positions corresponding to a cysteine residue, a substituted or non-substituted amino acid of a plurality of amino acid residues having no SH group is a vaccine composition, characterized in that failing to stabilize the change. In the first immunoglobulin, characterized in that the same region as the fragment of the second immunoglobulin molecule, except that the variant region, one or a plurality of amino acid is substituted in the variant region, the second immunoglobulin molecule is Wherein the at least some of the one or plural amino acid substitutions are substitutions of one or plural amino acid residues having no SH group at one or plural positions, wherein the position is disulfide in the second immunoglobulin molecule. One or multiple cysteine residues forming a bond, wherein one or multiple amino acid residues having no SH group at one or multiple positions corresponding to one or multiple cysteine residues forming a disulfide bond in the second immunoglobulin molecule Amino acid substitution, not substitution of, does not stabilize the change Vaccine composition, characterized in that.