KR20010015818A - Immunoglobulin molecules having a synthetic variable region and modified specificity - Google Patents

Abstract

The present invention provides a modified immunoglobulin molecule, in particular an antibody, wherein the antibody immunospecifically binds with one binding pair, wherein the immunoglobulin has a mutation region, wherein the mutation region is one or a plurality of complementary determinants. Wherein the crystal region has an amino acid sequence of the binding position for the member of the binding pair, the position is derived from another binding pair member, and is not found in the complementarity determining region in nature. The present invention further provides for the therapeutic and prophylactic use of modified immunoglobulins.

Description

Immunoglobulin molecule having synthesized variable moieties and modification specificity {IMMUNOGLOBULIN MOLECULES HAVING A SYNTHETIC VARIABLE REGION AND MODIFIED SPECIFICITY}

2. Background of the Invention

2.1. Antibodies and Immune System

Antibodies are proteins belonging to the immunoglobulin superfamily. Immunoglobulin superfamily includes T cell receptors, B cell receptors, cell-surface adsorption molecules such as co-receptor CD4, CD8, CD19, and non-denaturing domains of MHC molecules. In their soluble form, the antibody is one in which the glycoprotein produced by mature B cells called plasma cells is an antibody. Antibodies are secreted into the blood and other extracellular fluids, circulating throughout the body and responding to foreign antigens in all animals and humans.

Antibodies have two basic functions. The first is to recognize or bind foreign antigens. The second is to move other elements of the immune system, destroying foreign substances. Receptors on the surface of immune effector cells are designed to recognize antigens and cell surface markers on other cells. This cognitive process gives information about which markers are theirs and which are not theirs, which is an important factor involved in regulating the immune response to the antigen present.

The antigen portion to which the antibody binds is the antigenic determinant or epitope. Some antigens can induce an immune response, while others are recognized by the immune system itself. Antigens capable of inducing an immune response are called immunogens, which are large molecules having a molecular weight of at least 5000 Daltons, such as proteins, nucleic acids, carbohydrates, lipids. Smaller non-immunogenic molecules (called heptenes), when bound to large carrier molecules, can promote an immune response.

2.2. Structure of antibodies

The basic complete unit of the antibody is a four-chain Y-shaped structure (Fig. 1). In the early 1970s, Wu and Kabat gathered significant amounts of amino acid sequences from antibodies to describe their structure. In fact, every immunoglobulin superfamily is always partially and partially structured of four relatively conserved semi-rigid beta-sheets. Having three relatively short hypervariable sequence portions known as partial and conformity determining portions (CDRs) (Wu and Kabat, 1970. J. Exp. Med. 132 (2): 211-250; Wu and Kabat, 1971, Proc. Natl. Acad. Sci. US 4 68 (7): 1501-1506). This prediction was confirmed by crystal structure studies (Poljak et al., 1973, Proc Natl Acad Sci USA 70 (12): 3305-3310; Diesenhofer et al., 1976, Hoppe Seylers Z Physiol Chem (Germany) , West) 357 (10): 435-445: Diesenhofer et al., 1976, Hoppe Seylers Z Physiol Chem (Germany, West) 357 (10): 1421-1434).

Figure 1 shows the overall structure of the antibody. The antibody has a structure in which two short L chains are linked to two longer H chains by disulfide bonds. As can be seen in Figure 2, these H and L chain antibody proteins are composed of multiple domains, each of which consists of about 100 amino acid residues in length. Each L and H chain of the antibody has a variable portion at its amino acid terminus (each of which is referred to as V _L and V _H ); A variable portion of an antibody that confers antigen-binding specificity. The H chain variable domain and the L chain variable domain together form a single antigen-binding site, so the basic immunoglobulin unit has two antigen-binding sites.

The diversity in the variable portions of the L and H chains is limited to three "hypervariable" portions or CDRs. Each antibody molecule has a total of six CDRs, each of which contains about 5 to 10 amino acids and up to about 20 amino acids when endogenously recombined, which is common in certain antibody classes. . The three CDRs of the variable portion of each L and H chain form a loop, which are linked to each other and to the remaining four portions of the variable portion (called relatively conserved skeletal structures ("FRs") in the antibody molecule). . Diversity of antibodies is generally made by changing the sequence of CDRs.

The variable portion in each antibody is unique while the portion is always a highly conserved portion. The L chain always has only one partial domain, whereas the H chain consists of multiple domains, ie, domains called CH1, CH2, CH3 ... CHx. The partial domain always contains complementary binding, lymphocytes, granulocytes, unicellular lineage cells, binding to Fc receptors expressed by killer, stimulating B cells to stimulate proliferation of cells, blast cells, and other immune effector cells. It imparts the function of various antibody effect substances, such as. Other functioning functions include differentiation, activation of the complement cell lysis system, opsonization, and induction of macrophages. Antibodies of different isotypes have different always domains and therefore different effector functions. The best studied isotypes are IgG and IgM.

All animal species express several different kinds of antibodies. Based on structural differences, five antibody classes (IgG, IgA, IgM, IgD, IgB) and various subclasses have been identified within these classes, for example, the number of immunoglobulin units in a single antibody molecule, the number of individual units It is identified based on differences in structure, chain length and sequence of disulfide bridges. Although IgG is the most commonly used for pharmacological use for diagnosis and treatment to date, other types of antibodies may also be used for certain applications.

2.3. Antibody manipulation

The development of monoclonal antibody technology, first described by Kohler and Milstein (1975, Nature 256: 495-497), allows for the production of accurate, repeatable and infinitely unlimited antibodies. The Kohler and Milstein procedure is to fuse splenocytes obtained from immunized animals with immortalized myeloma cell lines to obtain hybridomas. Clones that produce antibodies with the required specificity are isolated from these hybridomas. Hybridomas make monoclonal antibodies homogeneous in their properties and specificities.

To date, the identification and production of suitable antibodies useful for diagnosis and treatment has been dependent on chance. The production of hybridomas that produce antibodies is by immunizing mice with antigens or by adding antigens to splenocyte preparations in vitro. Certain monoclonal antibodies with splenic cell populations and specificities have been dependent on the animal's immune response to antigen.

Another method of making antibodies that can be used for diagnosis and treatment has been developed as an alternative to the immunization process that requires the considerable labor mentioned above. One method is to clone antibody genes into phage virus that can express a single variable moiety on the virus surface (Clackson et al., 1991, Nature 352: 624; Marks et al., 1992, J. Mol. Biol. 222: 581; Zeedee et al., 1992, Proc. Natl. Acad. Sci. USA 39: 3175; Gram et al., 1992, Proc. Natl. Acad. Sci. USA 89: 3576). Phage library technology can be used to create many libraries that express significant amounts of intrinsic genetic diversity. However, such libraries are also limited by the antibody repertoire from which they are derived. Another approach is to mutate randomly and create large libraries using expressed variable domain genes (Pack (1997, High Quality Antibody Libraries, Abstracts of the Eighth International Conference of Antibody Engineering)). Both of these methods have been successful in making significant diversity, but generally fail to identify useful antibodies when compared to traditional immunization methods. This is because they make the CDR sequences random. In addition, the antibody produced through the immunization reaction of the mouse is limited to use in the treatment of humans. Since mouse monoclonal antibodies are foreign and thus immunogenic to humans, they induce human anti-mouse antibody (HAMA) responses (Shawler et al., 1985, J. Immunol. 135: 1530; Chatenaud et al. , 1986, J. Immunol. 137: 830.

2.4. Constraints that regulate intermolecular interactions.

The effect of pharmaceuticals is that certain physiological agents are useful for the prevention or alleviation of diseases, whereby pharmaceuticals enhance, antagonize, or mimic binding of one molecule to another, for example, binding of a ligand to a receptor, binding of a pathogen to a cellular receptor. And the ability to obtain pharmacological activity. To date, pharmaceuticals have been limited to unintentionally discovered natural or synthetic substances, and have been small molecule effectors that mimic binding to naturally occurring ligands. Although information relating to the structure of ligands or their binding sites is available, the methods currently available do not directly lead to the development of effective pharmaceuticals. Methods using molecular modeling to devise small molecule analogs based on crystal structure data for ligand-receptor binding pairs or screening for binding to receptors using peptide complex libraries or leading edge product extraction have not been proven to be reliable. In addition, such synthetic or natural products do not always have the ability to distinguish binding affinity and specificity for receptor subtypes, which may lead to unwanted side effects, due to insufficient criteria for pharmacological effects.

There is a need for ways to make or inhibit the effects of natural interactions more directly, and to design specific pharmaceuticals that can interact with specific binding pair members, which are closer to the behavior of naturally occurring ligands.

The literature mentioned herein should not be construed as an admission of the prior art of the present invention.

3. Summary of the Invention

The present invention is capable of implanting a binding site contained in another member of a binding pair to at least one CDR of an immunoglobulin molecule for one member of the binding pair, thereby imparting specificity to the immunoglobulin for the second member of the binding pair. It is based on the observation of the present inventors.

The present invention aims to design immunoglobulins, in particular antibodies, having certain specificities, wherein the method avoids the unpredictable immunization and screening processes currently used to isolate specific antibodies. In particular, a synthetic modified antibody capable of immunospecifically binding to one member of a binding pair is made, wherein the variable portion of the modified antibody has one or more CDRs comprising a binding sequence to a member of the binding pair, wherein the binding sequence is bound Derived from the other member of the pair. Therefore, such a method greatly simplifies the method of identifying an appropriate antibody, and can produce an antibody that can be used against many antigens that were inaccessible due to immune resistance and hidden expression.

Accordingly, the present invention provides a modified immunoglobulin, in particular an antibody capable of immunospecifically binding to a first member of a binding pair, wherein the binding pair consists of first and second members, and the antibody is comprised of the first of the binding pair. A variable domain having at least one CDR comprising the amino acid sequence of a binding site for a member, wherein the binding site is derived from a second member of the binding pair. In an appropriate aspect of the invention the amino acid sequence of the binding site is not found naturally in the CDRs.

The binding phase becomes any two molecules that can specifically interact with each other. In certain embodiments, the first member of a binding pair is a cancer antigen (eg, a molecule expressed on the surface of a cancer cell), an antigen of an infectious disease medium (eg, a molecule on the surface of an infectious disease medium) or a cell of an infectious disease medium. Become a receptor Such cancer antigens include human maintenance antigen (HMFG), epitopes of polymorphic epithelial mucosal antigen (PEM) or human colon carcinoma-associated protein antigens. Antigens of infectious disease media include Brambell receptor (FcRB), HSV-2 antigen, gonococcus, Treponema pallidum, Chlamydia trachomatis or human papillomavirus. . In another specific embodiment, the binding pair is a receptor-ligand binding pair, for example the first member of the binding pair is a bradykinin receptor.

The present invention provides methods for the treatment and prevention using the modified immunoglobulins of the present invention. For example, cancer or certain cancer antigens, antigens of an infectious disease medium, using modified antibodies comprising one or more CDRs having a binding site of a cancer antigen or an antigen of an infectious agent or a cell receptor of an infectious agent or Treatment and prevention of infectious diseases associated with expressing cellular receptors of an infectious disease medium.

The present invention also provides a method for screening or detecting or diagnosing a disease using the modified immunoglobulin of the present invention. For example, cancer or certain cancer antigens, antigens of an infectious disease medium, using modified antibodies comprising one or more CDRs having a binding site of a cancer antigen or an antigen of an infectious agent or a cell receptor of an infectious agent or Infectious diseases associated with expressing cellular receptors of an infectious disease medium can be screened, detected, and diagnosed.

The invention also provides therapeutic and diagnostic kits, pharmaceutical compositions comprising the modified immunoglobulins of the invention.

The invention also provides a method of producing a synthetically modified immunoglobulin.

Paragraph 6 describes the synthesis of synthesized modified antibodies having CDRs comprising the amino acid sequence of breadkinin comprising the binding sequence of the bradykinin receptor. The Examples demonstrate that such synthesized modified antibodies compete specifically with the Bredykinin by immunospecifically binding to the Bredykinin receptor and binding to the Bredykinin receptor. The activity of the synthesized modified antibodies is antagonized with antagonists of bradykinin activity.

Related Applications

This application claims the advantages of pre-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 a modified immunoglobulin molecule, more specifically an antibody that binds to one member of a binding pair and has at least one complementarity determining moiety (CDR) comprising the amino acid sequence of the binding site for a member of the binding pair. Related to. The present invention also provides a method of treating, diagnosing and screening a disease associated with the expression of a member of a binding pair using a modified antibody of the invention. The present invention provides pharmaceutical compositions and diagnostic kits comprising the modified antibodies of the invention.

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.

(A) in Figure 3A-C shows the structure of the expression vector pMRRO10.1, Kappa L chain of human always includes a 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 containing a sequence encoding the kappa always part of the L chain and the always part and hinge part of the H chain. For information on all these vectors, see Bebbington et al., 1991, Merhods in Enzymology 2: 136-145.

4A-H have amino acid and nucleotide sequences of the H and L chain variable domains with CDRs comprising the variable domain consensus sequence and the bradykinin sequence of the H and L chains of the synthetic antibody. All these sequences include the leader sequence. (A) shows the amino acid sequence of the consensus L chain variable portion ConVL1 and the corresponding nucleotide sequence. (B) shows amino acid and corresponding nucleotide sequence for L chain variable moiety BKCDR1 wherein CDR1 comprises a bradykinin sequence. (C) shows the amino acid sequence of the L chain variable moiety BDCDR2 wherein the CDR2 comprises a bradykinin sequence and the nucleotide sequence corresponding thereto. (D) shows the amino acid sequence of the L chain variable moiety BKCDR3 and its corresponding nucleotide sequence, wherein CDR3 comprises a bradykinin sequence. (E) amino acid sequence of the consensus L chain variable moiety ConVH1 and the corresponding nucleotide sequence. (F) CDR4 represents the amino acid sequence of the L chain variable moiety BKCDR4 comprising a bradykinin sequence and the corresponding nucleotide sequence. (G) CDR5 represents the amino acid sequence of the L chain variable moiety BKCDR5 comprising a bradykinin sequence and the corresponding nucleotide sequence. (H) CDR6 represents the amino acid sequence of the L chain variable moiety BKCDR6 comprising a bradykinin sequence and the corresponding nucleotide sequence.

Figure 5 illustrates the general steps of assembling an engineered gene encoding a synthesized modified antibody comprising the sequence of bradykinin. Oligonucleotides used for gene assembly are represented by "oligo1" to "oligo10".

(A) in FIGS. 6A and 6B show the nucleotide sequence of oligonucleotides used to assemble the bradykinin comprising the consensus L chain (ConVL1) and L chain variable moiety by the procedure shown in FIG. 5. (B) shows (A) the nucleotide sequence of the oligonucleotide used to assemble the bradykinin containing the consensus H chain (ConVH1) and the H chain variable moiety by the procedure shown in FIG. 5.

(A) in FIGS. 7A-C show that PGE ₂ synthesis is stimulated by bradykinin in SV-T2 cells, where the amount of PGE ₂ in each treatment uses ng per well. In the legend below the figure, "-" indicates culturing the cells without factor, and culturing the cells in the presence of "+" sms factor. The factor represents 1 nM Bradykinin (top row) or 1 nM HOE 140, Bradykinin antagonist (bottom row). (B) shows the stimulation of PGE ₂ synthesis by a specific synthesized modified antibody with a CDR comprising a bradykinin sequence, represented by pg / PGE ₂ , the synthetic antibodies BKCDR3 (square line), BKCDR4 (triangle line), BKCDR5 (Diamond line), consensus H chain variable moiety (circular line), media alone (open circular line). (C) bar graph shows PGE ₂ stimulation (PGE ₂ pg / well) in SV-T2 cells with or without bradykinin (represented by "+" or "-", respectively) Cultured with antibodies having a BKCDR3, BKCDR4, BKCDR5 variable domain or H chain consensus variable domain (ConVH).

The present invention relates to modified immunoglobulin molecules, particularly antibodies, which immunospecifically bind to the first member of a binding pair, which is determined by methods known in the art to measure the specific binding of the antibody to the antigens described in paragraph 5.7. Wherein the immunospecific binding excludes non-specific binding but does not need to be cross-active as seen in antibodies naturally occurring in the first member of the binding pair, and does not comprise the amino acid sequence of the second member of the binding pair. Having at least one complementarity determining moiety (CDR), wherein the amino acid sequence is the binding sequence of the first member of the binding pair. 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 antibody comprises a cancer antigen (molecule on the surface of a cancer or tumor cell), an infectious disease antigen (molecule on the surface of an infectious disease medium), a cell receptor for a pathogen, a receptor-ligand (preferably a receptor- A ligand or a ligand of a binding pair, wherein the ligand comprises a binding sequence to a cellular receptor for a receptor or ligand that binds to the receptor and induces a physiological response.

The invention also provides a method of treatment with the modified immunoglobulins of the invention, including, for example, antigens of a specific cancer antigen or infectious disease medium or antigens of an infectious disease medium or a binding sequence to a cell receptor of an infectious disease medium. By using a modified antibody comprising at least one CDR, an infectious disease medium is bound to a specific receptor to treat and prevent cancer or an infectious disease characterized by the presence of a specific antigen.

The invention also provides methods for diagnosis and screening using the modified immunoglobulins of the invention, e.g., against specific cancer antigens or infectious disease media antigens or infectious disease media antigens or infectious disease media cell receptors. A modified antibody comprising at least one CDR comprising a binding sequence can be used to detect cancer or an infectious disease characterized by the presence of a specific antigen by binding an infectious disease medium to a specific receptor.

For the sake of clarity, the detailed description of the present invention will be given in the following paragraphs but is not intended to be limited thereto.

5.1. Modified immunoglobulin molecule

The present invention relates to modified immunoglobulin molecules, particularly antibodies, which immunospecifically bind to the first member of a binding pair, which is determined by methods known in the art to measure the specific binding of the antibody to the antigens described in paragraph 5.7. Wherein at least one of the CDRs of the antibody comprises a binding site for 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 terms of appropriate invention the amino acid sequence of the binding site is not naturally found in the CDRs.

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 cancer antigens, infectious disease antigens, cell receptors for pathogens, cell receptors for receptors or ligands that participate in receptor-ligand binding pairs.

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 factors.

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 5.3.1), 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 invention, at least one CDR of the modified antibody comprises a binding site for antigen of an infectious disease medium (see description in paragraph 5.3.2.) Or a binding site of a cell receptor of an 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.

The modified immunoglobulin molecule of the invention can be any immunoglobulin, which is an unmodified-domain of a T cell receptor or B cell receptor, a cell expressing adsorbent molecule such as a co-receptor CD4, CD8, CD19 or MHC molecule. In a suitable embodiment, the immunoglobulin molecule is of the type of antibody molecule viscosity suitably selected from IgG, IgE, IgM, IgD, IgA, most suitably an IgG molecule. In another specific embodiment, the modified immunoglobulin molecule is a T cell receptor.

Immunoglobulins that are modified to make modified immunoglobulins are suitably monoclonal or synthetic antibodies to be any available immunoglobulin molecule. The antibody to be modified can be a naturally occurring or existing antibody and can be synthesized from a known antibody consensus such as the consensus sequence of the L and H chain variable moieties shown in Figs. 4A or B or any other antibody consensus or Germline (e.g., non-recombinant genomic sequence) (e.g., 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 a 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 abbreviated in Figure 2) are shown in Table 1.

Table 1

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.

In certain embodiments, the modified immunoglobulins of the present invention comprise a portion of a variable moiety, such as less than the skeletal moiety in the H chain or L chain, comprising three CDRs, wherein the variable moiety comprises one or two CDRs; Suitably between the framework structures.

In certain embodiments, the modified antibody immunospecifically binds to the bradykinin receptor (eg, but is not limited to, the modified antibody described in paragraph 6). In particular, embodiments provide modified antibodies to at least one CDRs comprising the amino acid sequence Arg-Pro-Pro-Gly-Phe-Gly-Phe-Ser-Pro-Phe-Arg.

In another specific embodiment, the modified antibody immunospecifically binds to a human milk antigen, and at least one of the CDRs of the modified antibody comprises an amino acid sequence selected from:

(Iii) Ala-Tyr-Trp-Ile-Glu;

(Ii) Glu-Ile-Leu-Pro-Gly-Ser-Asn-Asn-Ser-Arg-Tyr-Asn-Glu-Lys-Phe-Lys-Gly

(Iii) Ser-Glu-Asp-Ser-Ala-Val-Tyr-Tyr-Cys-Ser-Arg-Ser-Tyr-Asp-Phe-Ala-Trp-Phe-Ala-Tyr;

(Ⅳ) Lys-Ser-Ser-Gln-Ser-Leu-Leu-Tyr-Ser-Ser-Asn-Gln-Lys-Ile-Tyr-Leu-Ala

(Iii) Trp-Ala-Ser-Thr-Arg-Glu-Ser; and (iii) Gln-Gln-Tyr-Tyr-Arg-Tyr-Pro-Arg-Thr.

In a more specific embodiment, the CDRs of the H chain variable portion comprise the amino acid sequences (i)-(iii), while the CDRs of the variable portion of the L chain comprise the amino acid sequences of (iv)-(vi) above. Include.

In certain embodiments, the invention provides a modified antibody that binds to a human colon carcinoma-associated antigen and consists of a variable moiety having at least one CDR comprising one of the following amino acid sequences;

Thr-Ala-Lys-Ala-Ser-Gln-Ser-Val-Ser-Asn-Asp-Val-Ala;

Ile-Tyr-Tyr-Ala-Ser-Asn-Arg-Tyr-Thr;

Phe-Ala-Gln-Gln-Asp-Tyr-Ser-Ser-Pro-Leu-Thr;

Phe-Thr-Asn-Tyr-Gly-Met-Asn;

Ala-Gly-Trp-Ile-Asn-Thr-Tyr-Thr-Gly-Glu-Pro-Thr-Tyr-Ala-Asp-Asp-Phe-Lys-Gly;

Ala-Arg-Ala-Tyr-Tyr-Gly-Lys-Tyr-Phe-Asp-Tyr.

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. And other techniques known in the art)

Thus, in certain embodiments, modified antibodies have greater affinity or specificity for the antigen than naturally occurring antibodies that immunospecifically bind to the same antigen. In another embodiment, the modified antibody has a binding constant for an antigen of at least 2 × 10 ⁷ M.

Modified antibodies of the invention may further modify the antibody using methods known in the art, wherein the additional modifications should not prevent or inhibit the binding of the modified antibody to a particular antigen. In particular, the modified antibodies of the invention have one or more amino acid substitutions, deletions, insertions in addition to those inserted or substituted in the CDR sequences within the amino acid sequences of the binding sequences. Such amino acid substitutions, deletions or insertions may be any substitutions, deletions or insertions that do not interfere with or inhibit the immunospecific binding of the modified antibody to the target antigen. 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.

In addition, one or more amino acid residues in the sequence may be substituted with non-traditional amino acids or a chemical amino acid analog may be introduced by substitution or insertion into an immunoglobulin sequence. 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 certain embodiments of the invention, the modified immunoglobulin may be further modified to enhance its ability to induce an anti-idi response (which is 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). Such modifications remove or reduce disulfide bonds between or within the chain to reduce constraints due to the shape in the variable portion of the immunoglobulin. In particular, the modified immunoglobulins are further modified to replace one or more variable partial cysteine residues that make disulfide bonds with amino acid residues that do not contain an SH group.

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 involved in forming disulfide bonds can be identified by comparing the sequences with other antibody moieties known as disulfide bonds (e.g., the consensus sequences provided in FIGS. 4A and dp or Kabat et al., 1991m). sequences of Proteins of Immunological Interest 5th Ed., US Department of Health and Human Services, Bethesda, Maryland).

Table 1 provides the positions of cysteine residues that can form disulfide bonds in a large number of antibody molecules.

TABLE 2

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 responds 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. 4A. And residues such as those shown in the H and L chain variable subsequences described in E (i.e., determined by aligning side-by-side with the sequence of the H and L chain variable moiety or the consensus sequence as shown in FIG. 4A or E). Has meaning).

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 does not have an SH group or the residue at position 22 or 92 of the H chain has an SH group. Refers to an antibody substituted with an amino acid residue.

Amino acids that replace cysteine residues that form disulfide bonds are any amino acid residues that do not have an SH group, such as 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.

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 of the present invention, the present invention provides a functionally active fragment of a modified immunoglobulin, wherein the functionally active fragment is a fragment that is immunized when determined by any known method for measuring immunospecific binding. Specifically binding to the target antigen (described in 5.7). For example, such fragments include the F (ab ') ₂ , the L-chain always part, and the CH1 domain of the H-chain produced by pepsin treatment of antibody molecules, and are made by reducing the disulfide bridges of F (ab') ₂ . Fab fragments (FIG. 1; King et., 1992, Biochem, J. 281: 317) and Fv fragments, such as fragments comprising both variable partial domains of the H and L chains (Reichmann and Winter, 1988, J. Mol. Biol. 203: 825; King et al., 1993, Biochem. J. 290: 723), and the like.

The invention also encompasses single chain antibodies (SCAs) (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). Single chain antibodies are made by linking the H and L chain fragments of the Fv moiety by amino acid bridges. The present invention also provides H chain and L chain dimers and diabodies.

The invention also provides chimeric or humanized modified antibodies. 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 with the constant portion of human immunoglobulins. 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. In certain embodiments, the modified antibody synthesized is a chimeric antibody comprising the variable domain of a non-human antibody and the always domain of a human antibody.

In certain embodiments, modified antibodies of the invention are humanized antibodies, more suitably CDRs of the antibody (except for one or more CDRs having a binding sequence) are derived from non-human animals, skeletal parts and always parts Is derived from human antibodies (Winter et al. US Pat. 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 to antigen-respiratory conjugate viruses (Tempest et al. (1991, Bio-Technology 9: 267)).

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 variable region genes can be made using a variety of methods, such as site-directed mutagenesis, etc. in Jones et al., 1986, Nature 321: 522; Riechmann et al., 1988, Nature 332: 323; in vitro assembly of entire CDR-grafted variable regions (Queen et al., 1989, Proc. Natl. Acad. Sci. USA 86: 10029); and the use of PCR to synthesize CDR-grafted genes (Daugherty et al., 1991, Nucleic Acids Res. 19: 2471). CDRs-conjugated antibodies are made by conjugating the CDRs of murine monoclonal antibodies to the framework of human antibodies. After conjugation, it benefits from additional amino acid changes in the structural moiety to maintain affinity, because the framework residues are essential for maintaining the CDR shape, and some framework residues are known to be part of the antigen binding site. Thus, in certain embodiments of the invention, the modified antibody consists of variable moieties, wherein at least one of the frameworks has one or more amino acid residues different from the residues at positions in the naturally occurring framework structures.

In a suitable embodiment of the invention, the modified antibody is derived from a human monoclonal antibody. Complete human monoclonal antibodies can be made using transgenic mice. The mouse immunoglobulin gene position is replaced with the human immunoglobulin position provided in the in vivo affinity-maturation mechanism for producing human immunoglobulin.

In another embodiment, the present invention provides a fusion protein of the modified immunoglobulin of the invention (or a functionally active fragment thereof), for example, via covalent linkage of a modified immunoglobulin, which is not a 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 a suitable embodiment, the invention provides a modified immunoglobulin wherein IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, G-CSF, tumor Necrosis factor, porin, interferon-gamma, NK cell antigens, MHC-induced peptides and other parts of immune stimulating factors or covalently bound to cell growth factors.

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. For example, derivatives and analogues of the modified immunoglobulins of the present 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.

In certain embodiments of the invention, the modified immunoglobulin (or fragment thereof) is covalently linked to a therapeutic molecule to target the therapeutic molecule to a particular cell type or tissue, such as a tumor or cancer cell. The therapeutic molecule may be in the form of therapeutic molecules known in the art, for example chemotherapeutic agents, toxins such as lysine, antisense oligonucleotides, radionuclides, antibiotics, anti-viral or anti-parasitic agents and the like.

5.2. How to produce modified immunoglobulins

Modified immunoglobulins of the present invention can be produced by any method known in the art for immunoglobulin synthesis, in particular, produced by chemical synthesis or pre-expression, preferably produced by the synthetic expression technique.

Recombinant expression of the modified immunoglobulins, fragments thereof, of the present invention requires the construction of nucleic acids encoding modified immunoglobulins. Such isolated nucleic acids having nucleotide sequences encoding modified immunoglobulins can be prepared by any method known in the art, such as recombinant techniques or chemical synthesis (Creighton, 1983, "Proteins: Structures and Molecular Principles", WHFreeman). & Co., NY, pp. 34-49; and Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual.Cold Springs Harbor Press, NY) or encoded CDR sequences with binding sites PCR can be made in known immunoglobulin genes for processing nucleotide sequences.

Accordingly, the present invention provides a nucleic acid having a nucleotide sequence that encodes a modified immunoglobulin or functionally active fragment thereof of the present invention.

Preferably, the nucleic acid encoding a modified immunoglobulin is combined from a chemically synthesized oligonucleotide (Kmeier et al., 1994. Biotechniques 17: 242), which simply states that a portion of the sequence encoding the modified immunoglobulin is encoded. Synthesis of retained overlapping oligonucleotides, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

Accordingly, the present invention provides a method for producing a nucleic acid encoding a modified immunoglobulin, which method comprises (a) synthesizing a set of oligonucleotides, wherein said set encodes a portion of the nucleotide sequence encoding the artificial modified immunoglobulin. Oligonucleotides retained, consisting of oligonucleotides having a portion of the nucleotide sequence complementary to the nucleotide sequence encoding the modified immunoglobulin, each oligonucleotide having an overlapping terminal sequence with a different set of oligonucleotides, except that these oligonucleotides are artificial Has nucleotides encoding the N-terminus and C-terminus of the modified immunoglobulin; (b) allow oligonucleotides to hybridize or anneal to each other; (c) ligation of the hybridized nucleotides to produce nucleic acids with nucleotides encoding the artificial modified immunoglobulins.

Alternatively, a nucleic acid having a nucleotide sequence encoding modified immunoglobulin is made from an antibody molecule, or a nucleic acid having a nucleotide sequence encoding at least a variant region of an antibody molecule. Nucleic acid having a nucleotide sequence encoding an antibody molecule can be obtained from a clone of an existing antibody molecule or mutant domain or by isolation of a nucleic acid encoding the antibody molecule or mutant domain from an appropriate source, which source is preferably a cDNA library. For example, antibody DNA libraries or cDNAs made from cells or tissues expressing repeat regions of antibody molecules or artificial synthetic libraries (Clackson et al., 1991, Nature 352: 624; Hane et al., 1997, Proc Natl.Acad. Sci USA 94: 4937), these are made by hybridization with probes specific for specific antibody molecules or by PCR amplification with artificial primers that hybridize with the 3 'and 5' ends of the sequence.

Once the nucleic acid having the nucleotide sequence encoding at least the variant region of the antibody molecule is cloned, the binding site sequence can be inserted into the nucleotide sequence encoding one or more CDRs. Processing of this particular CDR coding sequence can be accomplished with routine recombinant DNA techniques known in the art. For example, a nucleotide sequence that encodes a CDR is replaced by a nucleotide that encodes a CDR having a specific binding position sequence, which is used for PCR-based methods and in vitro specific site mutagenesis. If a convenient restriction enzyme position is available for the nucleotide sequence of the CDR, the sequence can be cleaved with a restriction enzyme, and the nucleic acid fragment having the nucleotide sequence encoding the binding position can be ligated to the restriction position. A nucleic acid fragment having a binding position may be obtained from a nucleic acid encoding all or part of a protein having a binding position, or a sequence encoding the binding position and its reverse complement may be made from an artificial oligonucleotide having a binding position.

The nucleic acid encoding the modified antibody optionally retains the nucleotide sequence encoding the read sequence, which leads to secretion of the artificial antibody antibody.

When a nucleic acid encoding at least the mutant domain of a modified antibody is obtained, this inserts a region of the antibody into a vector containing the encoded nucleotide sequence (PCT Publication WO 86/05807; PCT Publication WO 89/01036; and US See Patent No. 5,122,464). Vectors having L or H chains for coexpression with nucleic acids that allow the expression of complete antibody molecules can be obtained and are known in the art, for example, pMRRO10.1, pGammal (Bebbington, 1991, Methods in Enzymology 2: 136-145.

The expression vector is then transferred to the host cell by conventional methods, and the transfected cells can be cultured by conventional techniques to make the antibodies of the invention. In particular, when a nucleic acid variant region of a modified antibody is made, the modified antibody can be expressed by the method exemplified in section 6 (see Bebbington, 1991, Methods in Enzymology 2: 136-145), for example modified immunoglobulin. Transfecting the expression vector encoded by COS cells temporarily, culturing the cells for a suitable period of time to express the immunoglobulin, and then collecting the supernatant of the COS cells, where the supernatant is secreted, expressing the immunoglobulin Hold.

The host cell used for expressing the recombinant antibody of the present invention is a bacterial cell such as Escherichia coli, in particular for the expression of recombinant antibody fragments, or preferably for the expression of eukaryotic cells, particularly recombinant antibody molecules. In particular, mammalian cells such as Chinese hamster ovary cells (CHO) or COS are used in combination with 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 antibody coding sequence of the present invention. Such host-expression systems represent carriers that produce and subsequently purify the coding sequence of interest, but also express cells in situ that express the antibody product of the invention when transformed or transfected with the appropriate nucleotide coding sequence. It may also be indicated. 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 the recombinant yeast expression vector carrying the antibody coding sequence; Insect cells infected with a recombinant virus expression vector (eg, baculovirus) containing an antibody coding sequence; Plant cell lines infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or recombinant plasmid expression vectors (e.g. Ti plasmid) carrying antibody coding sequences; 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, multiple expression vectors are preferentially selected according to the intended use for the antibody molecule to be expressed. For example, when producing such proteins in large quantities for the production of pharmaceutical compositions of antibodies, vectors which control the high level expression of fusion protein products that are readily purified are desirable. Such vectors include, for example, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2: 1791), wherein the antibody coding sequence is individually transferred to the vector frame through the lac Z coding region. Ligation results in fusion proteins: PIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13: 3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24: 5503-5509. Using pGEX vectors The foreign polypeptide is expressed as a fusion protein with glutathione S-transferase (GST) In general, these fusion proteins are water soluble and can be easily purified from lysed cells, where they are absorbed and the matrix glutathione-agarose Binding to beads and eluting in the presence of free glutathione The pGEX vector is designed to contain thrombin or factor Xa protease cleavage, allowing cloned target gene products to fall off 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. Antibody coding sequences are cloned into non-essential sites of the virus and placed under the control of an AcNPV promoter (eg, polyhedron promoter).

In mammalian host cells, a number of virus-based expression systems are used. When using adenovirus as an expression vector, the antibody coding sequence of interest is ligated to the adenovirus transcription / detoxification control complex (eg, post promoter, trilateral read sequence). This chimeric gene is subsequently inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (eg, the E1 or E2 region) results in a recombinant virus capable of surviving and expressing the antibody in the infected host (Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81: 355-359). Certain initiation signals are required for efficient translation of inserted antibody 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 lines stably expressing antibodies 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 antibodies. This processed cell line is particularly useful for the selection and evaluation of compounds that interact directly or indirectly with antibodies.

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 (Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1); When conferring resistance to hygromycin, hygro (Santerre et al., 1984, Gene 30: 147).

Expression levels of artificially modified antibodies 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. The two vectors carry the same selectivity mark which equally expresses the 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 before the H chain, avoiding excessive 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.

The present invention provides a recombinant cell having a vector encoding a phosphorus antigen, wherein the antibody has a CDR having an amino acid sequence of an active binding position from a binding pair member.

5.3. Therapeutic Uses of Artificial Modified Antibodies

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, but are not limited to, modified immunoglobulins of the present invention, functionally active fragments, analogs and derivatives thereof (see paragraph 5.1), nucleic acids encoding modified immunoglobulins of the present invention, and functionally active fragments thereof (paragraphs). (See 5.2).

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, pharmaceutical compositions containing modified antibodies (or functionally active fragments thereof) of the present invention immunospecifically bind to specific molecules, which treat or prevent diseases or disorders associated with the expression of specific molecules, eg, antigens. Can be used for In particular, in embodiments to be described in detail in the following subparagraphs, expression of specific antigens is carried out using immunospecific antibodies, antigens of infectious disease agents, modified antibodies that bind to cellular receptors for infectious disease agents. Prevention or treatment of related tumors, cancers or infectious diseases. Modified immunoglobulins that immunospecifically bind to ligands or receptors can be used to treat or prevent diseases associated with the absence or decrease of specific ligand receptor amounts. In certain embodiments, modified immunoglobulins can be used to treat or prevent autoimmune diseases, including, but not limited to, rheumatoid arthritis, lupus, ulcerative inflammation, and psoriasis. Modified immunoglobulins are also used to treat allergies.

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.

5.3.1. Cancer Treatment and Prevention

The present invention provides a method of treating or preventing cancer characterized by the presence of specific cancer antigens that are members of a binding pair. The method comprises administering to a subject in need of such treatment or prevention, a therapeutic agent of the invention, eg, an artificially modified antibody (or functionally active fragment thereof) that immunospecifically binds to a specific cancer antigen. It consists of a mutant domain with CDRs carrying amino acid sequences of binding sites for cancer antigens.

Cancer, including any disease characterized by neoplasia, tumors, metastases or unregulated cell growth, can be treated and prevented by administering the artificially modified antibodies of the present invention, wherein the modified antibodies are immunospecific with one or more antigens. And binds to the 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 paragraph 5.6, but are not limited to these examples.

For example, cancers and tumors associated with the following cancer and tumor antigens are treated or prevented by administering the phosphorus of the invention having sequences recognizing these cancer antigens on a CDR: KS1 / 4 pan-cancer (species) antigen ( 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 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 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, thioguanine , Urea hydroxide, cytarabine, cyclophosphamide, phosphamide, nitorourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, etoposide, campatin, bleomycin, doxorubicin, Idarubicin, daunorubicin, dactinomycin, pilicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, pasililitaxel, and docetaxel, but are limited to these examples I never do that. In suitable embodiments, the artificially modified antibody binds to a chemotherapeutic drug, another form of toxin (eg, lysine toxin, radionuclide), or any other drug that kills cancer or tumor cells or effectively stops cancer cell growth. In another suitable embodiment, the modified immunoglobulin has one CDR having a binding site for a cancer antigen, another CDR having a binding site for a surface molecule of an immune cell, and examples of such immune cells include T cells, B Although a cell, NK cell, K cell, TIL cell, or neutrophil is mentioned, It is not limited to these examples.

In certain embodiments of the invention wherein an amino acid sequence is included in the CDR of an artificially modified antibody, wherein the amino acid sequence immunospecifically binds to a colon cancer (species) -associated protein antigen, the antibody preferably has the following characteristics: (9) the antibody recognizes the epitooff of the protein component of the antigen but not the epitooff of the carbohydrate component of the antigen; (ii) the antigen is not detected in normal human tissue; (iii) The antigen is not detected in human cancer (species) cells except colon cancer cells. In another embodiment, the CDRs of the modified antibodies include amino acids that immunospecifically bind to the antigen, and the antigen is not detected in human cancers (species) except breast cancer (species) cells. In another embodiment, the CDRs of an artificially modified antibody include amino acids that immunospecifically bind to an antigen, and the antigen is not detected in human cancer (species) except ovarian cancer (species) cells.

5.3.1.1. 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, uterus, gastrointestinal tract, B lymphocytes or T lymphocytes. do. In another embodiment, treating and preventing sarcoma, melanoma or leukemia.

5.3.1.2. Precancerous disease

The therapeutic substance of the present invention is administered to treat precancerous diseases 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 release, increased glucose uptake, reduced 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 present invention is administered to a human patient to progress to cancer, melanoma or sarcoma of the ovary, breast, colon, lung, pancreas, skin, prostate, stomach, B lymphocytes, T lymphocytes or uterus To prevent.

5.3.2. Treatment of Infectious Diseases

The present invention also provides a method for preventing or treating an infectious disease by administering a therapeutic agent of the present invention, in particular an artificially modified immunoglobulin (or a functionally active fragment thereof), wherein the immunoglobulin molecule is used in the pathogenesis causing the infectious disease. Immunospecifically binds to an antigen, a cell receptor for an infectious disease agent, or an enzyme expressed by an infectious disease agent. As described below, infectious pathogens include, but are not limited to, viruses, bacteria, fungi, protozoa, and parasites.

In certain embodiments, the infectious disease provides a method of preventing or treating an infectious disease by administering a modified antibody (or functionally active fragment thereof) of an immunoglobulin of the present invention, wherein the modified antibody is Recognize one of the antigens specifically: 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), the 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 protein, parent protein or other proteins of dengue virus (Genbank accession no. M19197; Hahn et al., 1988, Virology 12: 17-180), measles virus hemagglutinin (Genbank accession no. M81899; Rota et al., 1992, Virology 188: 135-142), Herpes zoster virus type 2 glycoprotein gB (Gen bank 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 24M epitope (Beachey, 1985, Adv. Exp. Med. Biol. 185: 193), Gonococcal pilin (Rothbard and Schoolnik, 1985, Adv, Exp. Med. Biol. 185: 247), Pseudorabies virus g50 (gpD), Pseudorabies ) 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 53 protein.

Along with pathogens that bind to cell receptors, a list of cell receptors targeted for infectious disease treatment 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-II). ), 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-II), 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 (B virus), faxvirus are included, but are not limited to these.

Bacterial diseases that can be treated or prevented by the methods of the present invention include Mycobateria rickettsia, Mycoplasma, Neisseria species (eg, Neisseria mennigitidis, neisseria gonorrhoeae), Legionella, Shigella species spp.,), Vibrio cholerae, Streptococcus (e.g. Streptococcus pneumoniae), Cornebacteria diphtheriae, Clostridium tetani, Bordetella pertussis, Sea It is caused by bacteria including but not limited to Haemophilus spp., Chlamydia spp., And enterocoliogenic Escherichia coli.

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. In a suitable embodiment, the modified antibody is conjugated with a compound that is effective against the infectious disease agent targeted by the modified antibody, such compound being an antibiotic, an antifungal or an antiviral agent. In another suitable embodiment, the modified immunoglobulin consists of one CDR having a binding position to an antigen of an infectious disease pathogen, another CDR having a binding position to a surface molecule of an immune cell, for example T Although a cell, B cell, NK cell, K cell, or neutrophil is mentioned, It is not limited to these examples.

5.3. 3 gene therapy

In certain embodiments, a nucleic acid consisting of a sequence encoding an artificially modified antibody of the present invention is administered to treat or prevent a disease and condition associated with expression of a molecule to which the artificially modified antibody specifically binds.

In an embodiment of the invention, the therapeutic nucleic acid encodes a sequence that makes the modified immunoglobulin of the invention intracellular (without read sequence) or between cells (with lead sequence).

Any of the gene therapies available in the art can be used in accordance with the present invention. A typical method is given below.

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 one aspect, the therapeutic nucleic acid comprises an expression vector that expresses a modified immunoglobulin molecule in a suitable host. In particular, such nucleic acids have a promoter that selectively binds to the coding sequence for the modified phosphorus receptor, which promoter is induced or constructed and optionally tissue specific. In another embodiment, the modified antibody is expressed in the chromosome using nucleic acid flanking the region and region where the antibody coding sequence and any other desired sequence promote homologous recombination at a desired location on the genome (Koller and Smithies). , 1989, Proc. Nat'l.Acad.Sci.Usa 86: 8932-8935; Zijistra et al., 1989, Nature 342: 435-438.

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 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). 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.

5.3.4. Vaccine Immunization

Modified antibodies of the invention are used as vaccines in a subject, wherein immunization to the binding site of a particular molecule or antigen is preferred. The vaccines and methods of the invention are used to prevent a disease or to treat a particular disease or condition, wherein the anti-genetic response to a particular phosphorus agent is therapeutically or prophylactically useful.

The methods and compositions of the present invention are used in the induction of a humoral or cell-mediated response of a vaccine to a phosphorus agent in a subject. In certain embodiments, the methods and compositions induce a humoral response to a phosphorus airport administered in a subject. In another embodiment, the methods and compositions induce a cell-mediated response to a phosphorus airport administered in a subject. In another suitable embodiment, the methods and compositions induce both humoral and cell-mediated responses.

5.4. Pharmaceutical Compositions and Methods of Administration

5.4. 1. Composition and Administration

Therapeutic compositions containing modified immunoglobulins in the use according to the invention can be prepared by conventional methods using one or a plurality of physiologically acceptable carriers or excipients.

Thus, modified immunoglobulins (or functionally active fragments thereof, nucleic acids encoding antibodies), their physiologically acceptable salts, solvents are administered by inhalation or inhalation (through the mouth or nose), or oral, buccal, intestinal Can be made for external or rectal administration.

For oral administration, the therapeutic agents can take the form of tablets or capsules and include sintering agents (eg, sun gelatinized corn starch, polyvinylpyrrolidone, hydroxypropyl methylcellose); Filters (eg, lactose, microcrystalline cellulose or hydrogen phosphate); Lubricants (eg magnesium stearate, talc or silica); Refined digests (eg, potato starch or sodium starch glycolate); Or pharmaceutically acceptable excipients such as wetting agents (eg, sodium laurate sulfate). Liquid preparations for oral administration may take the form of solutions, syrups or suspensions, or they may be made of dry products which consist of water or other suitable carriers before use. Such liquid formulations may be used as suspensions (eg sorbitol syrup, cellulose derivatives or hydrogenated edible fats); Emulsifiers (eg, lectins or acacia); Non-aqueous carriers (eg almond oil, oily esters or sorted vegetable oils); Prepared by conventional methods using preservatives such as methyl or propyl-p-hydroxybenzoate or sorbic acid. The formulations may suitably include buffer salts, seasonings, colorants, sweeteners.

Preparations for oral administration are suitably prepared to provide controlled release of the active compound.

For oral administration, the therapeutic agents take the form of tablets or (lozenge) tablets prepared by conventional methods.

For inhalation administration, the therapeutic agent used in accordance with the present invention may be a pack or automatic, which maintains a constant pressure using a suitable oxidizing agent (e.g., dichlorodifluoromethane, trichlorodifluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). Easily delivered in the form of aerosol sprays from vending machines. In the case of an aerosol with a constant pressure, the dosage unit is determined by providing a valve that delivers the quantity in meters. Capsules or medicines of gelatin for use in inhalers or blowers are prepared to contain a mixture of the compound and a suitable powder such as lactose or sucrose.

The compounds are prepared for extra-intestinal administration by injection, for example by bolus injection or continuous infusion. Injectable compositions are prepared in unit dosage form, eg, in ampoules or in multidose containers, with an added preservative. The composition may take the form of a suspension, a solution, or an emulsion dissolved in an oily or water soluble carrier and contains a composition such as a suspension, stabilizer and / or diffusion agent. Alternatively, the active ingredient is present in powder form consisting of a suitable carrier, for example pyrogen-free sterile water, before use.

The therapeutic agent may be made of rectal compositions such as suppositories or retention enemas containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the compositions previously disclosed, the therapeutic agents may be made into storage agents. Such long acting compositions can be administered by infusion (eg, subcutaneously or intramuscularly) or by intramuscular injection. Thus, the compounds may be made with suitable copolymerizable or hydrophobic materials (such as oil-soluble emulsions) or ion exchange resins, or made into hardly degradable derivatives such as hardly degradable salts.

Modified immunoglobulins of the present invention are administered in a single composition with one or more antibodies that link individual compositions or conventional chemical or molecular biological methods. The diagnostic and therapeutic value of the antibodies of the invention is also extended by use in combination with radionuclides, toxins such as lysine, or chemotherapeutic agents such as methotrexate.

If desired, the composition may contain small amounts of wetting agents, emulsifiers, pH buffers. The composition may be a liquid solution, suspension, emulsion, tablet, pill, capsule, continuous release agent 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.

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 present invention in the form of complementary pharmaceutical carriers are also prepared, placed in suitable containers and labeled for the treatment of the indicated diseases.

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.

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.

5.4.2. Effective amount

Pharmaceutically effective amounts of compounds and nucleic acid sequences disclosed herein can be administered to a patient to treat certain diseases or conditions, such as cancer or infectious diseases. By therapeutically effective amount is meant a sufficient amount of the compound that has a beneficial effect on the health of the treated individual.

Toxicity and therapeutic efficacy of the compounds can be determined by determining standard methods in cell culture or experimental animals, such as LD ₅₀ (fatal dose of 50% population) and ED ₅₀ (effective dose in 50% population). The dose ratio between toxic and therapeutic effects is a therapeutic indicator and can be expressed as IC ₅₀ / ED ₅₀ . Compounds that exhibit toxic side effects can be used, in which case they will design a delivery system that targets the affected tissue location to minimize side effects by minimizing potential damage to uninfected cells.

Results from cell culture assays and animal studies can be used to create dosage ranges for human use. Doses of such compounds are in the range of purifying concentrations that include ED ₅₀ without toxicity. The dosage will vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods of the invention, the therapeutically effective amount can be initially assessed in cell culture assays. Single doses are prepared in animal models to achieve circulating plasma concentration ranges including IC50 as determined in cell culture. This information is used to more accurately determine the doses available in humans. Plasma levels can be measured by high performance liquid chromatography.

5.4.3. Vaccine Compositions and Administration

The present invention also provides a vaccine composition containing the therapeutic agent 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 immunoglobulins of the present invention are 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.

Various methods are used to introduce the vaccine compositions of the present invention, for example, through the oral, brain, transdermal, muscle, peritoneal, venous, subcutaneous, nasal routes or by oocyte method (upper bilateral needles). Scraping from) or other methods used for standard routes of the immunization process. In certain embodiments, egg methods have been used.

The exact dosage of the modified immunoglobulin molecule used in the preparation depends upon the characteristics of the patient when administered, which should be determined by the physician according to standard clinical techniques. The effective immunization amount should be an amount sufficient to create an immune response (eg, an anti-idiotype response) to the modified immunoglobulin in the host administering the vaccine preparation. Effective doses can be obtained by extrapolation from the pharmacokinetic response curve derived from the animal motel test.

5.5. Diagnostic method

Diagnosis and prognosis can be made using modified immunoglobulins, in particular antibodies (and functionally active fragments thereof), that bind to specific molecules that are members of a binding pair, as described herein. In various embodiments, the present invention provides for the measurement of binding pair members and the use of such measurements in clinical applications. Modified immunoglobulins of the present invention are used to detect antigens in biological samples, wherein the patient is tested for abnormally large numbers of molecules to which the modified immunoglobulins bind or for the presence of such abnormal forms of molecules. Abnormal levels ("aberrant levels") refer to increased or decreased levels relative to standard levels present in similar samples obtained from disease-free individuals or parts of the body. Modified antibodies of the invention can be included as reagents in kits used in diagnostic or prognostic techniques.

In certain embodiments of the invention, using a modified antibody of the invention that immunospecifically binds to a cancer or tumor antigen or infectious disease medium, a cancer, tumor or tumor associated with antigen expression of the cancer or tumor or infectious disease medium Diagnosis, prognosis or screening of infectious disease can be made. In a suitable aspect, the present invention provides a method for diagnosing, screening or predicting whether a cancer is characterized by an increased cancer antigen, wherein the cancer antigen is a first member of a binding pair consisting of a first member and a second member. Wherein the method consists in measuring the immunospecific binding level of the modified antibody in the subject to a sample derived from the subject, wherein the modified antibody immunospecifically binds to a cancer antigen, wherein the modified antibody comprises at least a second member of the second member. Consisting of a variable domain with a portion comprising one CDR, wherein the portion includes a binding site for a cancer antigen but is not naturally occurring in the CDR and is similar to that obtained in an individual without cancer or without cancer propensity. When compared with the level of immunospecific binding in the sample, the increase in the level of immunospecific binding indicates that cancer exists in the subject. Me or shows that have a tendency to get cancer.

In another suitable embodiment, the present invention provides a method for screening or diagnosing the presence of an infectious disease medium characterized by the presence of an antigen of an infectious disease agent, wherein the antigen consists of a first member and a second member. A first member of a binding pair, the method comprising measuring in the subject an immune specific binding level of the modified antibody to a sample derived from the individual, wherein the modified antibody immunospecifically binds to a cancer antigen, and the modified antibody Is composed of a variable domain having a portion comprising at least one CDR comprising at least four amino acid moieties of a second member, wherein the portion includes a binding site for a cancer antigen but is not naturally occurring in the CDR When compared with immunospecific binding levels in similar samples obtained from individuals without infectious diseases, Increased levels of immunospecific binding are indicative of the presence of an infectious disease medium in a subject.

In another suitable embodiment, the present invention provides a method for detecting the presence of abnormal levels of specific ligands or receptors in a subject derived sample, wherein the method comprises immunospecificity of the modified antibody to a sample having normal ligand or receptor levels. Modified antibodies that bind to specific ligands or receptors are made by comparing immunospecific binding to a sample.

Measuring molecules that bind to modified antibodies is valuable in detecting diseases associated with molecules in an individual, identifying the stage of the disease, screening for such diseases in a population, differential diagnosis, and under the physiological conditions of the individual. It is valuable to monitor the effectiveness of treatment in an individual.

The following test is designed to detect molecules to which the modified antibody is immunospecifically bound.

In certain embodiments, such diagnostic methods are used to detect abnormalities in gene expression levels or abnormalities in structures or abnormalities in tissue, cell or subcellular locations of specific molecules to be tested.

Tissues or cell types to be analyzed include those known to or likely to express particular molecules. Protein isolation methods used herein are as described in Harlow and Lane (Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). . Isolated cells can be derived from a patient or cell culture. Modified antibodies (or functionally active fragments thereof) useful in the present invention may further be used in histology, for example in immunofluorescence or immunoelectron microscopy to detect molecules in situ. In situ detection is performed by removing the histological specimen from the patient, for example by burying the diseased tissue in paraffin and providing the labeled modified antibody of the present invention. Modified antibodies (or functionally active fragments thereof) are used on a biological sample. If the molecule to which the antibody binds is present in the cytoplasm, it is desirable to make it penetrate into the cell and introduce the modified antibody into the cell. Through this process, it is possible to determine if a specific molecule is present and its distribution in the examined tissues. Using the present invention, one skilled in the art can modify any of any of a variety of histological methods (coloring process) to detect in situ.

Immune auditing of certain molecules generally involves incubating samples such as biological fluids, tissue extracts, freshly obtained cells or cultured cell lysates in the presence of detectably labeled modified antibodies, and which are not known in the art. Consisting of detecting the bound antibody.

The biological sample may be immobilized on a solid phase support or carrier such as nitrocellulose or other solid support capable of immobilizing cells, cell particles or soluble proteins. The support is then washed with appropriate buffer and treated with a detectable labeled modified antibody. The solid phase support is then washed twice with buffer to remove unbound antibody. The amount of label bound to the solid phase support can be measured using conventional means.

"Solid phase support or carrier" refers to any support capable of binding an antigen or an antibody. Known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified celluloses, polyacrylamides, gabbros, magnets and the like. The nature of the element may be to some extent soluble or insoluble for the purposes of the present invention. The support material may have any possible structural shape as long as the actual bound molecule can bind to the antigen or antibody. Thus, the shape of the support may be spherical, such as beads, cylindrical, such as the inner surface of the test tube or the outer surface of the rod. Alternatively, the surface may be flat, such as a sheet, a test strip, or the like. Proper support is polystyrene beads. Those skilled in the art will know many other suitable carriers capable of binding to antibodies or antigens and will be able to ascertain their suitability through routine experimentation.

The binding activity of a given modified antibody is determined using known methods. Those skilled in the art will be able to determine appropriate test conditions through routine experimentation.

One way to detectably label a modified antibody is to link it to the enzyme used in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2: 1-7, Microbiological Associates Quarterly Publication, Walkersville, MD; Voller et al., 1978, J. Clin. Pathol. 31: 507-520; Butler, 1981, Meth. Enzymol. 73: 482-523: Maggio. E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, FL ,; Ishikawa et al., (Eds,), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The enzyme bound to the modified antibody reacts with a suitable substrate, preferably a chromogenic substrate, to produce a chemical moiety that can be identified spectrophotometrically, fluorometrically, or visually. Enzymes used to detectably detect modified antibodies include malate dehydrogenase, staphylococcus nuclease, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, as Include, but are not limited to, paraginase, glucose oxidase, beta-galactosidase, ribonucleases, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholinesterase, and the like. I don't let you. Detection can be made using colorimetric methods using chromogenic substrates for enzymes. Compared to similarly prepared standards, the degree of enzymatic reaction in the substrate can also be visually compared and detected.

It can also be detected using a variety of different immunoassays. For example, synthetic antibodies or fragments are labeled with radioactivity to detect proteins using radioimmunoassays (Weintraub, 1986, Principles of Radioimmunoassays, Senventh Traning Course on Radioligand Assay Techniques, The Endocrine Society). Radioactive isotopes can be detected by means such as gamma counters, scintillation counters, or self-radiographs.

Modified antibodies can also be labeled with fluorescent compounds. Antibodies labeled with fluorescence can be identified by their fluorescence upon exposure to light of the appropriate wavelength. Among the most commonly used fluorescent labeling compounds are floresin isothiocyanate, rhodamine, phycoerytin, phycocyanin, allophycocyanin, o-phthalaldehyde and fluoroskamine.

Fluorescent metals such as ¹⁵² Eu or other lanthanamide series can be used to detectably label modified antibodies. Such metals are bound to the antibody using metal chelate groups such as diethylenetriaminepentaacetate (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Modified antibodies can be detectably labeled by binding to a chemiluminescent compound. The presence of chemiluminescent antibodies is determined by detecting the presence of luminescence that occurs during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, trometic acridium esters, imidazoles, acridium salts, oxalate esters and the like.

Similarly, bioluminescent compounds are used to label synthesized modified antibodies of the invention. Biological luminescence is a form of chemiluminescence found in the biological system that increases the effect of catalytic proteins on chemiluminescence reactions. The presence of a bioluminescent protein is determined by sensing luminescence. Important bioluminescent compounds for labeling purposes include luciferin, luciferase, aquiolin and the like.

5.6. Description of Therapeutic Uses

Therapeutic use of the present invention is tested in vitro and in vivo for the desired therapeutic or prophylactic activity before use in humans. For example, in vivo testing includes exposing appropriate cells taken from a cell line or cultured cell of an individual with a particular disease to a medicament or administering a therapeutic agent and observing the effect of the therapeutic agent on the cell.

Where the therapeutic agent is a modified immunoglobulin that recognizes cancer or tumor antigens, the possible effect of the modified immunoglobulin is to contact the therapeutic agent with cultured cells (taken from the patient or cultured cell line), and use known means for cell survival or Growth is examined and tested, where cell proliferation measures ³ H-thymidine binding, directly counts cells or changes in transcriptional activity of known genes such as proto-ongens such as fos, myc or cell cycle markers. Detect and inspect; Cell viability can be audited via trypan blue staining and differentiation can be visually confirmed based on morphological changes.

Where the therapeutic agent is a modified immunoglobulin that recognizes an infectious disease medium or a cellular receptor of an infectious disease medium, the possible effect of the antibody is to contact the therapeutic agent with cells infected from the infectious disease medium (taken from the patient or cultured cell line) and infectious disease Cells are examined for a decrease in media or a decrease in physiological indicators indicating infection of an infectious disease medium. Alternatively, the therapeutic agent is contacted with a cell that is susceptible to infectious medium but not actually infected with the infectious medium (taken from a cell line cultured or cultured in the patient), exposing the cell to the infectious medium, and The effect can be tested by confirming that the infection rate is lower than the infection rate of the cells that have not been contacted by the therapeutic agent. Infection of cells in an infectious disease medium can be examined by known methods.

If the therapeutic agent is a modified immunoglobulin specific for a particular ligand or receptor, the possible effect of the modified immunoglobulin is to contact the therapeutic agent with cells (taken from the patient or cultured cell line) expressing the receptor member of the binding pair and the therapeutic agent is the receptor. Determine whether the ligand interferes with binding or interferes with receptor signaling, or whether the therapeutic agent stimulates signaling of the receptor. Such indicators can be measured using any method known in the art for measuring ligand-receptor binding and receptor signal generation (see paragraph 6).

The therapeutic agent may be evaluated clinically in humans or its effectiveness is evaluated in appropriate animal models. The effect of a therapeutic agent may be colonized in any manner known in the art, after administration of the therapeutic agent to an animal model or human subject, and then evaluated for any indicator of the disease to be treated with the therapeutic agent in the animal or human. For example, the effectiveness of a therapeutic agent can be assessed by measuring the levels of molecules to which the modified antibody responds directly in the animal model or in humans, at appropriate times before, during and after treatment. It can be ascertained that there is no change or no change in the amount of molecules, which is related to the therapeutic effect in the individual. The level of the molecule can be determined using methods known in the art, for example 5.5. And the immunoassay methods in 5.7 can be used.

In another aspect, the modified antibody can test its effectiveness by monitoring whether an individual has a recovery or amelioration of the disease from a particular disease associated with the molecule to which the synthesized modified antibody is opposed. When the modified antibody is associated with a tumor or cancer antigen, the specific tumor or cancer progression is carried out according to known diagnostic or screening methods for monitoring the cancer or tumor. For example, cancer or tumor progression can be monitored by examining the level of a particular cancer or tumor antigen (or another antigen associated with a particular cancer or tumor) in an individual's serum or by injecting labeled antibodies specific for the antigen. have. In addition, other imaging techniques, such as CT scan or Sonograp or other imaging methods, can be used to monitor the progression of the cancer or tumor. Biopsies may be performed. In humans, prior to making such an attempt, it is desirable to test the effect of the modified immunoglobulin in animal models with certain cancers or tumors.

If the therapeutic agent is specific for an antigen of an infectious disease medium or an antigen of a cell receptor of an infectious disease medium, the effect of the modified antibody is to administer the modified antibody to the individual (human or animal with a disease), and the level or symptom of the specific infectious disease. Monitor and do this. Moisture of an infectious disease medium is checked by any method known in the art, for example, to monitor the level of an infectious disease medium, for example in the case of viruses, viral titers, bacterial titers are monitored. The level of infectious disease medium is determined by measuring the level of antigen to which the modified immunoglobulin responds. Reduction of infectious disease medium levels or alleviation of symptoms of infectious disease medium indicates that the modified antibody is effective.

When the therapeutic agent is administered as a vaccine, the immune effect of the vaccine composition comprising the modified antibody of the invention is determined by monitoring the anti-idiotype response of the test animal after immunization with the vaccine. The development of a humoral response indicates that there is a systemic immune response, and in particular other components of the cell-mediated immune response are important for protection against disease. Test animals may include mice, rabbits, chimpanzees, and real humans. The vaccine made in the present invention infects chimpanzees for experiments. However, because chimpanzees are protected species, antibody responses to the vaccines of the present invention are actually studied in smaller, less expensive animals, so that one or two optimal immunoglobulin molecules or optimal ones that can be effectively used in chimpanzee studies. Identify immunoglobulin complex molecules.

The immune response in the test subject is tested in a variety of ways, such as by testing using known techniques, such as responsiveness of the generated immune serum to the antibody and attenuation of signs of disease in the immunized host or protection from infection. Examples include enzyme linked immunosorbent assay (ELISA), immunoblot, radioimmunoprecipitation and the like.

As an example of a suitable animal test, the vaccine compositions of the present invention are tested in rabbits to determine their ability to induce anti-idiotype responses to modified immunoglobulin molecules. For example, New Zealand White rabbit males are grown with specific-pathogen-free (SPF). Each test population of rabbits receives an effective amount of vaccine. The reference rabbit population was injected with vaccine Tris-HCl pH 9.0 1 mM containing naturally occurring antibodies. Blood is drawn every week or two, serum is analyzed for anti-idiotype antibodies against modified immunoglobulin molecules, and for anti-anti-idiotype antibodies in which the modified antibodies are specific for the reactive ninety antigens. Test (radioimmunoassay; Abbott Laboratories). The presence of anti-idiotype antibodies was examined using ELISA. It is beneficial to test vaccines in mice because rabbits provide a variety of responses due to their various growth characteristics.

5.7. Testing of Modified Immunoglobulins

After constructing an immunoglobulin having one or more CDRs comprising a binding site for a particular molecule, binding between the resulting modified antibody and the particular molecule is assessed using any binding assay known in the art. Such testing can be performed to select antibodies that exhibit greater affinity or specificity for a particular antigen.

For example, various immunoassays known in the art can be used to test the binding of modified antibodies to specific molecules. Examples of methods that may be used include radioimmunoassay, ELISA (enzyme linked immunosorbent assay), " Sandwich "immunoassay, immunoradioactivity audit, gel diffusion precipitation reaction, immunodiffusion assay, in situ immunoassay (using colloidal gold, enzyme or radioisotope labels), western blot, precipitation reaction, aggregation test (e.g., Gel aggregation test, hematoglutinin test), complement complement test, immunofluorescence test, Protein A, immunoelectrophoresis test. In one embodiment, the antibody is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody can be detected by detecting the binding of a secondary antibody or reagent to the primary antibody. In another embodiment, the secondary antibody may be labeled. Many methods used to detect binding in immunoassays are known and are also within the scope of the present invention.

In vitro assay systems useful for evaluating the binding of modified antibodies to a target molecule are described below. In brief, the two components interact with each other (e.g., bind to each other to form a temporary but complex, and incubate the reaction mixture of the modified antibody and the test sample for a sufficient time, wherein the complex is removed from the reaction mixture. Such testing can be performed in a variety of ways, for example, one method of performing such testing is to immobilize a solid phase modified antibody or test substance, and at the end of the reaction, immobilized antibody / In one embodiment of such a method, the modified antibody can be labeled directly or indirectly, and the test sample is immobilized on a solid surface In practice, it is convenient to use microtiter plates in solid form. The immobilized component can be fixed by non-covalent or covalent adhesion. Covalent adhesion is achieved by simply coating a solid sample with a test sample solution and drying it.

To run the test, an unfixed component is added to the coated surface comprising the fixed component. After the reaction is complete, the unreacted components are removed under conditions in which any complex formed may remain fixed in the solid phase. Complexes immobilized on a solid phase can be detected in a variety of ways. In the case of labeling existing unfixed components in advance, detection of a label immobilized on the surface indicates that a complex has been formed. In the case where existing unfixed components have not been previously labeled, indirect labels can be used to detect complexes immobilized on the surface.

Alternatively, the reaction may be carried out in the liquid phase, where the reaction product is separated from the unreacted components and detects the complex.

5.8. Transgenic animals

The invention also provides an animal that has been transformed (including nucleic acid encoding a modified immunoglobulin) to a modified immunoglobulin (or a fragment having functional activity thereof) of the invention. Animals of any species, including, but not limited to, mice, rats, rabbits, guinea pigs, sheep, pigs, micro-pigs, goats, etc., may be viewed using non-human primates (eg, baboons, monkeys, chimpanzees). The transgenic of the invention can make an animal.

Thus, in certain embodiments, the invention provides a non-recombinant animal comprising a recombinant nucleic acid comprising a nucleotide sequence encoding a modified immunoglobulin of the invention, in particular immunospecifically binding to a cancer or tumor antigen To a nucleic acid encoding a modified antibody that provides a non-recombinant human animal transfected with a nucleic acid encoding a modified antibody or that immunospecifically binds to an antigen of an infectious disease medium or a cellular receptor of an infectious disease medium. Animals that are not recombinant humans that have been transfected are provided.

Antibody transgenes are introduced into the animal using any technique known in the art to create an established line of transgenic animals. Such techniques include pronuclear microinjection (Hoppe and Wagner, 1989, U.S. Pat. No. 4,873,191); Intermediary retrovirus mediated gene delivery into germline (Van der Putten et al., 1985, Proc. Natl. Acad. Sci. USA 82: 6148-6152); Gene targeting to embryonic liver cells (Thompson et al., 1989, Cell 56: 313-321); Electroporation of the embryo (Lo, 1983, Mol Cell. Biol. 3: 1803-1814); Sperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57: 717-723), and such techniques are described in Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol. See 115: 171-229.

The present invention provides transgenic animals that carry nucleotide sequences encoding antibodies transfected with these transgenes into these cells and animals that transfer the transgene to some but not all of these cells, such as mosaic animals. The transgenes may be combined in a single transgene or concatamer such as a head-head arrangement or a head-tail arrangement. Transgenes can be selectively activated by introducing them into specific cell types (Lasko et al. (Lasko et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6232-6236). The regulatory sequences required for cell type-specific activation will depend on the particular cell type of interest, which is known to those skilled in the art. Gene targeting is preferred when the nucleotides encoding the synthesized antibody transgene are inserted into the chromosomal region of the endogenous immunoglobulin. In short, when such a technique is used, a vector comprising some nucleotide sequences homologous to endogenous immunoglobulins is inserted into the nucleotide sequence of the endogenous immunoglobulin gene via homologous recombination with the chromosomal gene, thereby functioning of this sequence. It is designed for insertion in order to destroy it. The transgene can optionally be introduced into a particular cell type to inactivate endogenous immunoglobulins only in that cell type (Gu et al. (Gu et al., 1994, Science 265: 103-106)). The regulatory sequences required for such cell-type specific inactivation depend on the particular cell type desired and are known to those skilled in the art.

Methods of making single-copy transgenic animals bound to selected sites are known in the art (eg, Bronson et al., 1996, Proc. Natl. Acad. Sci. USA 93: 9067-9072).

Once a transgenic animal is made, expression of the recombinant antibody gene is tested using standard techniques. PCR techniques are used to initially screen using Southern blot analysis and to analyze animal tissue to confirm that the transgene has been inserted. Techniques used to identify mRNA levels of transgenes in the tissues of transgenic animals include, but are not limited to, Northern blot analysis, in situ hybridization reaction analysis, and RT-PCR of tissue samples obtained from animals. Immunohistochemical evaluation is performed using antibodies specific for antibody transgene products in tissue samples expressing genes.

6. Examples; Synthesized Modified Antibodies Including Bradykinin

In this embodiment, the construction of a synthesized modified antibody that immunospecifically binds to the bradykinin receptor (BR) will be described. The bradykinin receptor binds to a unique ligand called bradykinin. BR-bradykinin interaction is one example of a binding pair used in the methods of the invention. BR-bradykinin interactions occur when amino acids in the bradykinin, ie amino acids known as binding sites, contact the bradykinin receptors. The modified antibody synthesized in this example includes an amino acid derived from a bradykinin binding site. Thus, such synthesized modified antibodies can mimic the bradykinin ligand and predict binding to the bradykinin receptor (BR). Six synthesized modified antibodies comprising a bradykinin sequence are constructed and binding to BR is described as follows.

The method for producing a synthesized modified antibody comprising the bradykinin binding sequence can be represented as follows;

1) using an oligonucleotide to manipulate the variable region gene to have a bradykinin binding sequence and a CDR;

2) the engineered variable region gene is then inserted into a mammalian expression vector having the appropriate always portion;

3) infecting mammalian cells with the L and H chains and expressing the synthesized modified antibody; And

4) Examine the modified antibodies synthesized for BR binding.

6.1. Construction of Variable Partial Genes Including Bradykinin Binding Sites

In order to construct a variable region gene encoding a CDR including a bradykinin binding site, the following strategy is carried out.

First, the single stranded oligonucleotides are annealed to create double stranded DNA fragments with overhanging ends (as diagramed in Figure 5, Step 1; see also, Kutemeier et al., 1994 BioTechniques 17: 242). In particular, using automated technology from GenoSys Biotech Inc, oligonucleotides of liquid 80 bases in length corresponding to the sequence of interest with overlapping portions of 20 bases are synthesized. Specific sequences of such oligonucleotides are shown in FIGS. 6A and 6B (to construct variable portions of the L and H chains, respectively). 6A shows oligonucleotide sequences used to engineer L chain variable partial genes comprising a bradykinin binding sequence. 6B shows an oligonucleotide sequence used to construct an H chain variable partial gene comprising a bradykinin binding sequence. Oligo complexes and two consensus moieties (ConVL1 and ConVH1) used to manipulate the six bradykinin CDRs (BKCDR1, BKCDR2, BKCDR3, BKCDR4, BKCDR5, BKCDR6) are shown in Table 5 below.

Table 5

In order to complex oligos to the desired genes, variable partial genes are engineered as described below using 10 or 12 oligo groups. Each oligonucleotide is 5 'phosphorylated as follows; 25 μl of each oligo is incubated at 37 ° C. for 1 hour in the presence of T4 polynucleotide kinase, 50 mM ATP. The reaction is stopped by heating at 70 ° C. for 5 minutes and then ethanol precipitates. Once phosphorylated, complementary oligonucleotides (oligo1 + oligo10, oligo2 + oligo9, oligo3 + oligo8, oligo4 + oligo7, oligo5 + oligo6) as shown in Figure 5 were mixed in a sterile source centrifuge tube and mixed for 5 minutes. The tubes are annealed by heating in a water bath at 65 ° C. and cooled in Ceylon for 30 minutes. Annealing results in short double stranded DNA with adhesive ends.

Double stranded DNA fragments with adhesive ends are then ligated to longer strands (FIGS. 5, steps 2-4) to allow the assembled variable partial genes to be assembled. In particular, DNA fragments with adhesive ends are ligated in a water bath (maintained at 16 ° C.) for 2 hours in the presence of T4 DNA ligase and 10 mM ATP. Annealed oligo 1/10 is mixed with annealed oligo2 / 9 and annealed oligo3 / 8 is mixed with annealed oligo4 / 7. The resulting oligos are labeled oligo1 / 10/2/9 and oligo3 / 8/4/7. Oligo3 / 8/4/7 is then ligated to oligo5 / 6. The resulting oligo3 / 8/4/7/5/6 is ligated to oligo1 / 10/2/9 to create a full length variable partial gene.

Alternatively, when using 12 oligos populations, the order of addition is indicated by oligo number; 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 = 1/12/2 / 11,3 / 10 + 4/9 = 3/10/4 / 9,5 / 8 + 6/7 = 5/8/6 / 7,1 / 12/2/11 + 3/10/4/9 = 1/12/2/11/3/10/4 / 9,1 / 12 / 2/11/3/10/4/9 + 5/8/6/7 = full length variable partial genes. Eight variable partial genes are constructed in this manner. Four genes become L chain variable moieties and four genes become H chain variable moieties. Engineered L chain genes include ConVL1, ie, the consensus L chain variable moiety without a bradykinin sequence; An L chain variable moiety comprising a bradykinin sequence in BKCDR1, ie, CDR1; An L chain variable moiety comprising a bradykinin sequence in BKCDR2, ie CDR2; LK variable region comprising a bradykinin sequence in BKCDR3, ie CDR3. Engineered H chain genes include ConVH1, ie, the consensus H chain variable region without a bradykinin sequence; An H chain variable moiety comprising a bradykinin sequence in BKCDR4, ie CDR4; An H chain variable moiety comprising a bradykinin sequence in BKCDR5, ie CDR5; BKCDR6, ie, an H chain variable moiety comprising a bradykinin sequence in CDR6. The sequences of eight engineered variable partial genes are shown in FIGS. 4A-4F.

Each engineered gene resulting from a combination of oligonucleotides is processed as follows;

The resulting engineered variable partial genes are purified via gel electrophoresis. To remove unligated excess oligonucleotides and other incomplete DNA fragments, the ligated product is placed on a 1% low melt agarose gel at 110V for 2 hours. The major band containing the full length DNA product is cut out and placed in a sterile 1.5 ml centrifuge tube. To release DNA from agarose, the gel digest is cleaved with f3-AgraseI at 40 ° C. for 3 hours. Precipitate DNA with 0.3 M MaOAc and isopropanol at -20 ° C for 1 hour and centrifuge at 12,000 rpm for 15 minutes. Purified DNA pellet is resuspended in TE buffer, 50 [mu] l of pH 8.0. The engineered variable partial genes are amplified using PCR. In particular, 100 ng of the engineered variable region gene is mixed in buffer with 25 mM dNTPs, 200 ng primer, 5 U high fidelity thermostable Pfu DNA polymerase. DNA is amplified 28 times. The resulting PCR product was analyzed on 1% agarose gel.

Each purified DNA corresponding to the engineered variable partial gene is subsequently inserted into the pUC19 bacterial vector. pUC19 is an E. coli plasmid vector containing 54 base pair polylinkers in lacZ and Amp selectivity markers with 2686 base pairs. To prepare the vector for incorporation of the engineered variable partial gene, 10 μg pUC19 was linearized with HincI1 (50 U) at 37 ° C. for 3 hours, with blunt ends and 5 ′ GTC. To prevent self-religion, the linear vector DNA is treated with 25U calf visceral alkaline phosphatase (CIP) for 1 hour at 37 ° C. to eliminate phosphorylation. About 0.5 μg diphosphorylated linear vector DNA is mixed with 3 μg phosphorylated variable partial gene to insert the engineered variable partial gene into the pUC19 vector, at 16 ° C., in the presence of T4 DNA ligase (1000U). Incubate for 20 hours.

Bacterial cells are transformed using bacterial vectors containing the engineered variable partial genes. In particular, freshly prepared competent DH5-α cells, 50 μl, were mixed with 1 μg pUC19 containing the engineered variable region genes and transferred to electroporation cubec (0.2 cm gap; Bio-Rad). Each cubic is pulsed with a Bio-Rad Gene Pulser at 2.5kV / 200ohm / 25μF. Then immediately 1 ml SOC medium is added to each cubic and cells are recovered in centrifuge tubes at 37 ° C. for 1 hour. One release of cells from each transformation is removed, diluted 1: 100, and 100 μl is plated on LB plates containing ampicillin (Amp 40 μg / ml). Each plate is incubated overnight at 37 ° C. due to the presence of Amp markers. Only transformants containing the pUC19 vector can be grown in LB / Amp medium.

One transformed colony is selected and grown overnight with constant stirring at 37 ° C. in a 3 ml LB / Amp sterile glass tube. Plasmid DNA is isolated using Easy Prep columns (Pharmacia Biotech) and suspended in 100 μl TE buffer, pH 7.5. To confirm the presence of the gene insert in pUC19, 25 μl plasmid DNA in each colony was treated with HincI1 restriction endonuclease for 1 hour at 37 ° C. and analyzed on a 1% agarose gel. The plasmid DNA containing the gene inserted in this way loses the restriction site (5 '.. GTCGAC..3'), so that the enzyme is not cleaved and transferred to a completely closed (CC) DNA on the gel and without an insert. The plasmid is cleaved and transferred to linear (L) double stranded DNA fragments.

In order to identify the correct gene sequence of the engineered variable region gene, and to eliminate the possibility of unwanted mutations occurring during the construction process, DNA sequencing was performed in M13 / pUC reversed phase (5'AACAGCTATGACCATG) in an automated ABI 377 DNA Sequencer. 3 ') is used as a clone and the PCR gene product is made using a 5' terminal 20 crab salt primer (5 'GAATTCATGGCTTG GGTGTG 3'). All clones were found to contain the correct sequence.

Six engineered variable partial genes comprising a bradykinin sequence can be made via this example method. Shown in Table 6 shows the names of the modified antibodies synthesized in the variable partial genes and the positions corresponding to the bradykinin binding sequences. For example, a synthesized antibody, that is, an antibody named hAbBKCDR1, includes a bradykinin binding sequence (BK) in CDR1 of the variable partial L chain gene (V _L ). Such synthesized antibodies have a consensus sequence (con) in the variable partial H chain gene (V _H ).

Table 6

The amino acid sequences corresponding to the variable portions of each of the six synthesized modified antibodies of this example are shown in Table 7. CDRs are shown in bold. The bradykinin binding amino acid is ArgProProGlyPheSerProPheArg, which is underlined in the CDRs. Table 5 also describes the consensus sequences of human kappa L chain V _L subunit and human H chain V _H subunit I gene. If the consensus CDRs are too short to contain the complete bradykinin binding site sequence, the amino terminus of the bradykinin binding site is missing, since the carboxy terminal residues are known to be more intermediate to receptor binding. (Stewart and Vavrek, Chemistry of peptide B2 bradykinin antagonists, pp. 5196, Burch, RM, editor, Bradykinin Antagonists, Basic and Clinical Research, New York: Marcel Dekker, 1991; hereby incorporated by reference).

TABLE 7

6.2. Insertion of engineered variable genes into mammalian expression vectors

A complete antibody L chain contains both variable and always portions. The H chain of an intact antibody includes a variable portion, always a portion and a hinge portion. In order to construct complete L and H chains, the modified variable region gene is inserted into a vector containing the appropriate always part. An engineered variable region gene having a bradykinin sequence inserted into the L chain CDRs is inserted into the pMRRO10.1 vector (FIG. 3A), which contains the human kappa L chain always part. Insertion of the engineered L chain variable portion into this vector provides a complete L chain sequence. Or an engineered variable partial gene having a bradykinin sequence inserted into the H chain CDR is inserted into the pGAMMA1 vector (FIG. 3B), which vectorene human IgG1 always comprises a partial and cognitive partial sequence. Insertion of the engineered H chain variable partial gene into such a vector has a complete H chain sequence.

To manipulate a mammalian vector encoding a complete antibody, the complete H chain and L chain sequences are inserted into a single mammalian expression vector (Bebbington, CR, 1991, in METHODS: Methods in Enzymology, vol. 2, pp. 136-145). The generated vector encodes both L and H chains of the antibody, which is called pNEPuDGV (FIG. 3C).

6.3. Expression of Modified Antibodies Synthesized in Mammalian Cells

To confirm production of the assembled antibody, the pNEPuDGV vector is transfected into COS cells. COS cells (transformed into the African monkey kidney cell line, CV-1, origin-deleted SV40 virus) are used to temporarily express the synthesized antibody for a short time, because of cloning a circular plasmid containing the origin of replication. Because of the ability to make a very large number of copies. Antibody expression vectors are transfected with COS7 cells (obtained from the American Type Culture Collection) using calcium immobilization (Sullivan et al., FEBS Lett. 285: 120-123). Transfected cells are incubated for 72 hours in Dulbecco's modified Eagle's medium to obtain supernatants with antibodies comprising bradykinin. Supernatants of transfected COS cells were examined using an ELISA method for assembled IgG. The ELISA method is to collect samples and standards on 96-well plates coated with anti-human IgG Fc. Bound assembled IgG is detected with anti-human kappa chains and substrate tertamethylbenzidine (TMB) linked to horseradish peroxidase (HRP). Color development is proportional to the amount of assembled antibody in the sample.

6.4. Synthetic modified antibodies comprising bradykinin bind to the bradykinin receptor by mimicking the bradykinin ligand.

It can be predicted that the synthesized modified antibody engineered to include the bradykinin binding sequence binds the bradykinin receptor (BR) by mimicking the bradykinin ligand. To confirm that such synthesized modified antibodies bind to BR, the synthetic antibodies are tested in a bradykinin receptor binding assay. Testing of synthetic antibody binding to BR is carried out in the following manner. SV-T2 cells are transformed into fibroblasts that express about 3,000 bradykinin receptors (BR) per cell. Stimulation of bradykinin receptors in SV-T2 cells rapidly increases PGE2 synthesis in proportion to bradykinin binding. Thus, PGE2 released into the medium exhibits receptor binding.

As can be seen in FIG. 7A, PGE2 synthesis is stimulated about 4 times more by adding 1 nM bradykinin (ligand). PGE2 synthesis can be quantified by ELISA. Receptor antagonist HOE-140 was tested in FIG. 7A. Adding HOE-140 and bradykinin or HOE-140 does not induce PGE2 synthesis.

In addition, expressed modified antibodies as seen in FIG. 7B were tested for their ability to bind and stimulate bradykinin receptors. The bradykinin receptor is stimulated in SV-T2 cells using a medium of COS cells transfected with hABBKCDR3, hABBKCDR4, hABBKCDR5, or the antibody expression vector pNEPuDGV1 encoding the consensus. Synthetic antibodies with variable chain portions BKCDR3 and BKCDR5 stimulated PGE2 synthesis in a dose dependent manner. BKCDR4, ConVH medium alone, HOE-140 did not stimulate PGE2 synthesis (FIG. 7B). The cells exposed to BKCDR4 lack PGE2 synthesis, indicating that the CDR4 consensus sequence was too short to accommodate the entire bradykinin binding sequence. Table 6 compares the consensus CDR amino acid sequences and the BKCDR sequences. The modified modified antibodies BKCDR3 and BKCDR5 are described as having complete receptor binding to the native ligand bradykinin. As can be seen in FIG. 7C, the addition of bradykinin promotes PGE2 synthesis by about four times (second bar on the left). The addition of BKCDR3 or BKCDR5 to cells previously stimulated with native bradykinin inhibits PGE2 synthesis stimulated by bradykinin.

Table 8

Together these results indicate that a modified antibody comprising a bradykinin binding site can bind to a bradykinin receptor.

The invention is not limited to the specific embodiments described herein. In addition, those skilled in the art will recognize that various modifications of the invention are possible in addition to those described herein. Such modifications also fall within the scope of the appended claims.

The various references mentioned herein are incorporated by reference.

Claims (122)

In a modified immunoglobulin that immunospecifically binds to a binding pair of a first member, the binding pair consists of a first member and a second member, and the immunoglobulin comprises one or more CDRs retaining a partial region of the second member. And a variant region having a binding site for the first member, but not found in the CDRs in nature, wherein the first member is a cancer antigen. The modified immunoglobulin according to claim 1, which is an antibody. The modified immunoglobulin of claim 1, wherein the first member is a tumor antigen. 4. The modified immunoglobulin of claim 3, wherein the tumor antigen is a polymorphic epithelial intact antigen. 4. The modified immunoglobulin of claim 3, wherein the tumor antigen is human colon cancer (species) -associated protein antigen. The method of claim 5, wherein the region is Thr-Ala-Lys-Ala-Ser-Gln-Ser-Val-Ser-Asn-Asp-Val-Ala, Ile-Tyr-Tyr-Ala-Ser-Asn-Arg-Tyr -Thr, Phe-Ala-Gln-Gln-Asp-Tyr- Ser-Ser-Pro-Leu-Thr, Phe-Thr-Asn-Tyr-Gly-Met-Asn, Ala-Gly-Trp-Ile-Asn-Thr -Selected from Tyr-Thr-Gly-Glu-Pro-Thr-Tyr-Ala-Asp-Asp-Phe-Lys-Gly, Ala-Arg-Ala-Tyr-Tyr-Gly- Lys-Tyr-Phe-Asp-Tyr Modified immunoglobulins characterized by having an amino acid sequence. 4. The modified immunoglobulin of claim 3, wherein the tumor antigen is a human colon cancer (species) -associated protein antigen. 4. The modified immunoglobulin of claim 3, wherein the tumor antigen is a human milk fat sphere antigen. The method of claim 8, wherein the region is Ala-Tyr-Trp-Ile-Glu, Glu-Ile-Leu-Pro-Gly- Ser-Asn-Asn-Ser-Arg-Tyr-Asn-Glu-Lys-Phe-Lys -Gly, Ser-Glu-Asp-Ser-Ala-Val-Tyr- Tyr-Cys-Ser-Arg-Ser-Tyr-Asp-Phe-Ala-Trp-Phe-Ala-Tyr, Lys-Ser-Ser-Gln -Ser-Leu- Leu-Tyr-Ser-Ser-Asn-Gln-Lys-Ile-Tyr-Leu-Ala, Trp-Ala-Ser-Thr-Arg-Glu-Ser, Gln-Gln-Tyr-Tyr-Arg Modified immunoglobulin having an amino acid sequence selected from -Tyr-Pro-Arg-Thr. Ala-Tyr-Trp-Ile-Glu, Glu-Ile-Leu-Pro-Gly- Ser-Asn-Asn-Ser-Arg-Tyr-Asn-Glu-Lys-Phe-Lys- Gly, Ser-Glu-Asp-Ser-Ala-Val-Tyr- Tyr-Cys-Ser-Arg-Ser-Tyr-Asp-Phe-Ala-Trp-Phe-Ala-Tyr, Lys-Ser-Ser-Gln- Ser-Leu- Leu-Tyr-Ser-Ser-Asn-Gln-Lys-Ile-Tyr-Leu-Ala, Trp-Ala-Ser-Thr-Arg-Glu-Ser, Gln-Gln-Tyr-Tyr-Arg- A modified immunoglobulin comprising a second CDR having a portion of a second member having an amino acid sequence selected from Tyr-Pro-Arg-Thr. 4. The modified immunoglobulin of claim 3, wherein the tumor antigen is an antigen for cancer of the breast, ovary, uterus, prostate, bladder, lung, skin, colon, pancreas, gastrointestinal tract, B lymphocytes or T lymphocytes. The method of claim 1, wherein the cancer 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, paragraph Prostate Specific Membrane Antigen, Fetal Carcinoma Antigen, Polymorphic Endothelial Antigen, Human Fatty Acid Antigen, Colorectal Tumor-Related Antigen 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, angiogenesis Tumor 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 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 peptides. In a modified immunoglobulin that immunospecifically binds to a binding pair of a first member, the binding pair consists of a first member and a second member, and the immunoglobulin comprises one or more CDRs retaining a partial region of the second member. Wherein said region retains a binding site for the first member, but is not found in the CDRs in nature, and the first member is an antigen of an infectious disease pathogen. The modified immunoglobulin according to claim 13, which is an antibody. 14. The modified immunoglobulin of claim 13, wherein the infectious etiology is a bacterium. 14. The modified immunoglobulin of claim 13, wherein the infectious etiology is a viral child. 14. The modified immunoglobulin of claim 13, wherein the infectious etiology is a parasitic bacterium. The method of claim 13, wherein the antigen for the infectious disease pathogen is a Brambell receptor, HSV-2 antigen, antigen of Gonococcus, antigen of Treponema Pallidum, antigen of Chlamydia trachomatis, Or modified immunoglobulin, characterized in that it is selected from the antigens of human Papillomavirus. The method of claim 13, wherein the antigen for the infectious disease pathogen is influenza virus hemagglutinin, human respiratory syncytial virus G glycoprotein, deep protein of dengue virus, parent protein of dengue virus, measles virus hemagglutinin, monocytic herpes. Virus type 2 glycoprotein gB, poliovirus I VP1, envelope glycoprotein of HIV I, hepatitis B surface antigen, diphtheria toxin, Streptococcus 24M epitope, Gonococcal pilin, Pseudora 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, Sulfurina hydodysenteriae protective antigen, bovine viral diarrhea glycoprotein 55, Newcastle disease Irus hemagglutinin-neuuraminidase, swine cold hemagglutinin, swine cold neouraminidase, infectious bovine nasal bronchitis virus glycoprotein E, infectious laryngitis virus glycoprotein G or glycoprotein I, lacrose ( La Crosse) Glycoprotein, Fetal Diarrhea Virus, Hepatitis B Virus Deep Protein, Hepatitis B Virus Surface Antigen, Equine Influenza Virus Type A / Alaska 91 Neuuraminidase, Equine Influenza Virus Type A / Miami 63 New Urami Nidase, Equine Influenza Virus Type A / Kentucky 81 New Uramidase, 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 Polymorphic Virus Nucleocapsid Protein, Bovine Parainfluenza Virus Type 3 Fusion Protein , Bovine parainfluenza virus type 3 hemagglutinin New woorami The modified immunoglobulin according to claim, the bovine viral diarrhea virus glycoprotein 48, is selected from bovine diarrhea virus per 53 protein. 16. The infectious disease agent according to claim 15, wherein the infectious disease pathogen is Mycobateria rickettsia, Mycoplasma, Neisseria spp., Legionella, Shigella spp., Vibrio cholera. (Vibrio cholerae), Streptococci, Cornebacteria diphtheriae, Clostridium tetani, Bordetella pertussis, Haemophilus spp., Chlamydia Strain (Chlamydia spp.,), Escherichia coli (Escherichia coli). The method of claim 16, wherein the infectious disease pathogens are hepatitis A, hepatitis B, hepatitis C, influenza, chickenpox, adenovirus, herpes simplex virus type I, herpes simplex type II, right wing, nasal cold virus, echovirus, rota Virus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, hantan virus, coxsackie virus, mumps virus, measles virus, rubella virus, poliovirus, human immunodeficiency Virus type I, human immunodeficiency virus type II, picornavirus, enterovirus, calciviridia, norwalk virus group, togavirus, alphavirus, flavivirus, coronavirus, laviz virus, marburg virus, Ebola virus, parainfluenza virus, orthomyxovirus, field virus , Arenavirus, leo virus, 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 Modified immunoglobulin, characterized in that it is selected from human herpesvirus-6, monkey herpesvirus 1, and faxvirus. 18. The modified immunoglobulin according to claim 17, wherein the infectious disease agent is selected from malaria, eymeria, resimania, coccidioea, trimansomoma, and fungus. In a modified immunoglobulin that immunospecifically binds to a binding pair of a first member, the binding pair consists of a first member and a second member, and the immunoglobulin comprises one or more CDRs retaining a partial region of the second member. Wherein the region does not have an Asn-Ala-Asn-Pro or Asn-Val-Asp-Pro sequence, and is not found in CDRs in nature, and the first member is responsible for infectious disease pathogenesis. Modified immunoglobulin, characterized in that the intracellular receptor for. 24. The modified immunoglobulin according to claim 23, which is an antibody. 24. The modified immunoglobulin of claim 23, wherein the infectious disease pathogen is a bacterium. 24. The modified immunoglobulin of claim 23, wherein the infectious disease agent is a virus. 24. The modified immunoglobulin of claim 23, wherein the infectious disease pathogen is a parasite. 24. The cellular receptor of claim 23, wherein the cellular receptor is an LPV receptor, adenylfp silylase, 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, immune Globulin 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-acetylgal Lactosamine-low 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 Modified immunoglobulin, characterized in that selected from beta receptors. In a modified immunoglobulin that immunospecifically binds to a ligand-receptor binding pair of a first member, the binding pair consists of a first member and a second member, wherein the immunoglobulin has a partial region of the second member. 17. A modified immunoglobulin comprising a variant region having a CDR above, wherein said region retains a binding position for the first member but is not found in the CDRs in its natural state. The modified immunoglobulin of claim 29, wherein the antibody is an antibody. 30. The modified immunoglobulin of claim 29, wherein the first member is a receptor. The modified immunoglobulin of claim 29, wherein the first member is a ligand. The modified immunoglobulin of claim 29, wherein the first member is a receptor agonist. The modified immunoglobulin of claim 29, wherein the first member is a receptor antagonist. 30. The modified immunoglobulin of claim 29, wherein the first member is a bradkinin ligand. 36. The modified immunoglobulin of claim 35, wherein the region consists of an Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg amino acid sequence. 32. The receptor of claim 31, wherein the receptor is an opioid receptor, glucose transporter, glutamate receptor, ophanin receptor, erythropoietin receptor receptor, insulin receptor, lx leucine kinase receptor, KIT granulocyte-colonized stimulator receptor, somatropin receptor, Modified immunoglobulin, characterized in that selected from glial-induced neurotropic factor receptor, gp39 receptor, G-protein receptor type, β2-adrenergic receptor. 31. The ligand of claim 30, wherein the ligand is cholecystokinin, galanin, IL-1, IL-2, IL-4, IL-5, IL-6, IL-11, chemotin, leptin, protease, neuropeptide Y, new cow Lokinin-1, Neuurokinin-2, Neuurokinin-3, Bambecin, Gastrin, Colticotropin-releasing hormone, Endothelin, Melatonin, Somatostatin, Vasodilatory visceral peptide, Epidermal growth factor, Tumor necrosis factor, Modified immunoglobulin, characterized in that selected from dopamine, endothelin. 31. The modified immunoglobulin of claim 2, 14, 24 or 30 wherein the antibody is selected from IgG, IgE, IgM, IgD, IgA. 31. The modified immunoglobulin of claim 2, 14, 24 or 30, wherein the fragment is capable of immunospecific binding to the first member. The modified immunoglobulin of claim 40, wherein the fragment is selected from Fab, (Fab ′) 2, H chain dimers, L chain dimers, Fv fragments. 31. The modified immunoglobulin of claim 2, 14, 24 or 30, wherein the region is insertion in a CDR. 31. The modified immunoglobulin of claim 2, 14, 24 or 30, wherein the region replaces one or multiple amino acids of the CDRs. 31. The modified immunoglobulin of claim 2, 14, 24 or 30 wherein said CDRs carrying said region are germline CDRs. 31. The modified immunoglobulin of claim 2, 14, 24 or 30, wherein the CDRs carrying the region are non-germline CDRs. 31. The modified immunoglobulin of claim 2, 14, 24 or 30, wherein the region is at least 4 amino acids. 31. The modified immunoglobulin of claim 2, 14, 24 or 30, wherein the region is between 10-20 amino acids. 31. The modified immunoglobulin according to claim 2, 14, 24 or 30, wherein the CDR having the region has about 25 amino acids. 31. The modified immunoglobulin of claim 2, 14, 24 or 30 wherein the CDRs carrying the region are the first CDRs of the H chain variant region. 31. The modified immunoglobulin of claim 2, 14, 24 or 30 wherein the CDRs carrying the region are the second CDRs of the H chain variant region. 31. The modified immunoglobulin of claim 2, 14, 24 or 30 wherein the CDRs carrying the region are the third CDRs of the H chain variant region. 31. The modified immunoglobulin of claim 2, 14, 24 or 30 wherein the CDRs carrying the region are the first CDRs of the L chain variant region. 31. The modified immunoglobulin of claim 2, 14, 24 or 30 wherein the CDRs carrying the region are the second CDRs of the H chain variant region. 31. The modified immunoglobulin of claim 2, 14, 24 or 30 wherein the CDRs carrying the region are the third CDRs of the H chain variant region. 31. The modified immunoglobulin of claim 2, 14, 24 or 30, wherein at least one CDR has a binding site region. 31. The modified immunoglobulin of claim 2, 14, 24 or 30, wherein the second CDR has a second binding position to a molecule other than the first member. 59. The modified immunoglobulin of claim 56, wherein the molecule other than the member is a molecule located on the surface of an immune cell. 58. The modified immunoglobulin of claim 57, wherein the immune cell is T cell, B cell, NK cell, K cell, TIL cell or neutrophil. 31. The modified immunoglobulin according to claim 2, 14, 24 or 30, characterized by much higher specificity for one member than a naturally occurring antibody that immunospecifically binds to the first member. 31. The modified immunoglobulin according to claim 2, 14, 24 or 30, which has a much higher affinity for one member than a naturally occurring antibody that immunospecifically binds to the first member. 31. The modified immunoglobulin according to claim 2, 14, 24 or 30, characterized by showing a binding constant of at least 2 × 10 ⁷ M for the first member. The modified immunoglobulin of claim 2, 14, 24 or 30, wherein the antibody has an affinity constant of at least 2 × 10 ⁷ M for the first member. 30. The modification according to claim 1, 13, 23 or 29, wherein one or more cysteine residues are substituted with one or more amino acid residues having no SH group in the variant region of the immunoglobulin forming a disulfide bond. Immunoglobulins. 64. The modified immunoglobulin of claim 63, wherein at least one of the one or more cysteine residues is at position 23 or 88 of the L chain variant region. 64. The modified immunoglobulin of claim 63, wherein at least one of the one or more cysteine residues is at position 23 or 92 of the H chain variant region. 64. The modified immunoglobulin of claim 63, wherein at least one of the amino acid residues having no SH group is alanine. In a molecule consisting of a variant domain that immunospecifically binds to a binding pair of a first member, the binding pair consists of a first member and a second member, and the mutation domain comprises one or more regions having a partial region of the second member. Wherein said region retains a binding site for the first member, but is not found in the CDRs in nature, and the first member is a cancer antigen. In a molecule consisting of a variant domain that immunospecifically binds to a binding pair of a first member, the binding pair consists of a first member and a second member, and the mutation domain comprises one or more regions having a partial region of the second member. A molecule having a CDR, not found in the CDP in nature, wherein the region has a binding site for a first member, wherein the first member is an antigen of an infectious disease agent. In a molecule consisting of a variant domain that immunospecifically binds to a binding pair of a first member, the binding pair consists of a first member and a second member, and the mutation domain comprises one or more regions having a partial region of the second member. Having a CDR, the region has a binding position for a first member whose binding position does not have an ASn-Ala-Asn-Pro or Asn-Val-Asp-Pro sequence, and is not found in the CDRs in nature; Wherein said first member is a cellular receptor for infectious disease pathogen. In a molecule consisting of a mutant domain that immunospecifically binds to a ligand-receptor binding pair of a first member, the binding pair consists of a first member and a second member, and the mutation domain comprises a partial region of the second member. Wherein said region has one or more CDRs retained, said region retains the binding site for said first member but is not found in the CDRs in nature. The molecule of claim 67, 68, 69, or 70, wherein the molecule is a single chain antibody. 70. The molecule of claim 67, 68, 69, or 70 further comprising a certain domain. 73. The molecule of claim 72, wherein the mutant domain is obtained from a mouse antibody, except for the region-containing CDRs, wherein the region is obtained from a human antibody. The molecule of claim 72, wherein the mutant domain has a framework region obtained from a human antibody and a CDR obtained from a mouse, except for the region-containing CDRs, wherein the region is obtained from a human antibody. 75. The molecule of claim 74, wherein the variant region has a framework region in which one or more amino acid changes have occurred, in terms of naturally occurring framework regions. 70. The molecule of claim 67, 68, 69, or 70, wherein the molecule is fused with an immunostimulatory factor, growth enhancing factor, or functional fragment thereof via covalent bonds. 77. The method of claim 76, wherein the immunostimulatory factors are interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-10, interleukin-12, interleukin-15, G-colon stimulator, tumor necrosis Molecule, selected from factor, porin, interferon-gamma, NK cell antigen. An isolated nucleic acid comprising the nucleotide sequence encoding the modified immunoglobulin of claim 1, 13, 23, or 29. An isolated nucleic acid comprising a nucleotide sequence encoding the molecules of claims 67, 68, 69, and 70. 79. The isolated nucleic acid of claim 78, wherein the nucleic acid is a vector. 80. The isolated nucleic acid of claim 79, wherein the nucleic acid is a vector. 79. The isolated nucleic acid of claim 78, wherein the nucleic acid is recombinant. 80. The isolated nucleic acid of claim 79, wherein the nucleic acid is recombinant. A recombinant non-human animal comprising the nucleic acid of claim 78, A recombinant non-human animal comprising the nucleic acid of claim 79, A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of the modified immunoglobulin of claim 1, 13, 23 or 29 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising a therapeutic or prophylactically effective amount of a modified immunoglobulin of claim 67, 68, 69 or 70 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of the nucleic acid of claim 78 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of the nucleic acid of claim 79 and a pharmaceutically acceptable carrier. 83. A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of the recombinant cell of claim 82 and a pharmaceutically acceptable carrier. 84. A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of the recombinant cell of claim 83 and a pharmaceutically acceptable carrier. A vaccine composition comprising a carrier sufficient for inducing an immune response of the modified immunoglobulin of claim 1, 13, 23 or 29 and a pharmaceutically acceptable carrier. A vaccine composition, comprising a carrier that is sufficient to induce an immune response of a molecule of claims 67, 68, 69 or 70 and a pharmaceutically acceptable carrier. 64. A vaccine composition comprising a carrier sufficient for inducing an immune response of the modified immunoglobulin of claim 63 and a pharmaceutically acceptable carrier. In a method of identifying, measuring or detecting a cancer antigen in a sample to be tested, the cancer antigen is a first member of a binding pair consisting of a first member and a second member, wherein the method (a) contacting a sample to be tested with a modified immunoglobulin capable of immunospecifically binding to a cancer antigen under conditions in which the modified immunoglobulin can immunospecifically bind to any cancer antigen in the sample, wherein the modified The immunoglobulin consists of a variable domain having at least one CDR comprising a portion of the second member, wherein the portion of the second member comprises a binding portion to a cancer antigen that does not occur naturally in the CDR; (b) detects whether any binding of modified immunoglobulins to cancer antigens has occurred, Wherein if there is a binding of the modified immunoglobulin to the cancer antigen, the method comprises that the cancer antigen is present in the sample. In a method of identifying, measuring or detecting an antigen of an infectious disease medium in a sample to be tested, the antigen is a first member of a binding pair consisting of a first member and a second member, wherein the method (a) contacting a sample to be tested with a modified immunoglobulin capable of immunospecifically binding an antigen under conditions in which the modified immunoglobulin can immunospecifically bind any antigen in the sample, wherein the modified immunoglobulin Is composed of a variable domain having at least one CDR comprising a portion of the second member, wherein the portion of the second member comprises a binding portion to an antigen that does not naturally occur in the CDR; (b) detect if any binding of the modified immunoglobulin to the antigen has occurred and Wherein if there is a binding of the modified immunoglobulin to the antigen, the method comprises that the antigen is present in the sample. In a method for identifying, measuring or sensing a ligand in a sample to be tested, the ligand is a first member of a binding pair consisting of a first member and a second member, wherein the method (a) contacting a sample to be tested with a modified immunoglobulin capable of immunospecifically binding a ligand under conditions in which the modified immunoglobulin can immunospecifically bind any ligand in the sample, wherein the modified immunoglobulin Is composed of a variable domain having at least one CDR comprising a portion of the second member, wherein the portion of the second member comprises a binding portion to a ligand that does not occur naturally in the CDR; (b) detect if any binding of the modified immunoglobulin to the ligand occurred and Wherein the binding of the modified immunoglobulin to the ligand is indicative of the presence of the ligand in the sample. In a method of identifying, measuring or sensing a receptor in a sample to be tested, the receptor is a first member of a binding pair consisting of a first member and a second member, wherein the method (a) contacting a sample to be tested with a modified immunoglobulin capable of immunospecifically binding to a receptor under conditions in which the modified immunoglobulin may immunospecifically bind to any receptor in the sample, wherein the modified immunoglobulin Is composed of a variable domain having at least one CDR comprising a portion of the second member, wherein the portion of the second member comprises a binding portion to a receptor that does not occur naturally in the CDR; (b) detect if any binding of the modified immunoglobulin to the receptor has occurred and Wherein if there is a binding of the modified immunoglobulin to the receptor, the method comprises the presence of the receptor in the sample. In a kit for detecting a cancer antigen, the cancer antigen is a first member of a binding pair consisting of a first member and a second member, and the container of the kit (a) modified immunoglobulins capable of immunospecifically binding to cancer antigens; In this case, the immunoglobulin is composed of a variable part having at least one CDR including a part of the second member, wherein a part of the second part is a part including a binding site to a cancer antigen not found in nature in the CDR, And (b) a kit comprising means for detecting the binding of a cancer antigen to an immunoglobulin. In a kit for detecting an antigen of an infectious disease medium, the antigen is a first member of a binding pair consisting of a first member and a second member, (a) modified immunoglobulins capable of immunospecifically binding to an antigen; Wherein the immunoglobulin consists of a variable portion having at least one CDR comprising a portion of the second member, wherein a portion of the second portion is a portion comprising a binding site to an antigen not found in nature in the CDR, and (b) a kit comprising means for detecting the binding of an antigen to an immunoglobulin. In a kit for detecting a cell receptor of an infectious disease medium, the cell receptor is a first member of a binding pair consisting of a first member and a second member, (a) modified immunoglobulins capable of immunospecifically binding to cellular receptors; Wherein the immunoglobulin consists of a variable portion having at least one CDR comprising a portion of the second member, wherein a portion of the second portion is not found in nature in the CDR and the sequence is Asn-Ala-Asn-Pro or A portion comprising a binding site to a cell receptor other than Asn-Val-Asp-Pro, and (b) a kit comprising means for sensing the binding of cellular receptors to immunoglobulins. In a kit for detecting a ligand, the ligand is a first member of a binding pair consisting of a first member and a second member, (a) modified immunoglobulins capable of immunospecifically binding to ligands; Wherein the immunoglobulin consists of a variable portion having at least one CDR comprising a portion of the second member, wherein a portion of the second portion is a portion comprising a binding site to a ligand that is not found in nature in the CDR, and (b) a kit comprising means for sensing the binding of a ligand to an immunoglobulin. In a kit for detecting a receptor, the receptor is a first member of a binding pair consisting of a first member and a second member, (a) modified immunoglobulins capable of immunospecifically binding to receptors; Wherein the immunoglobulin consists of a variable portion having at least one CDR comprising a portion of the second member, wherein the portion of the second portion is a portion comprising a binding site to a receptor not found in nature in the CDR, and (b) a kit comprising means for sensing the binding of the receptor to an immunoglobulin. In a method of diagnosing cancer or screening for the presence of cancer or predicting the cancer occurrence, wherein the cancer antigen is a first member of a binding pair consisting of a first member and a second member, wherein the cancer antigen is increased. Way The immunospecific binding level of the modified immunoglobulin to the sample derived from the subject is measured in the subject, wherein the modified immunoglobulin immunospecifically binds to the cancer antigen and the modified immunoglobulin comprises a portion of the second member. Consisting of a variable domain having at least one CDR, wherein a portion has a binding site to a cancer antigen that does not occur naturally in the CDR; Increased levels of immunospecific binding compared to immunospecific binding levels in similar samples obtained from individuals with no cancer or no cancer propensity indicate that the cancer is present or prone to cancer. Way. In a method for diagnosing or screening an infectious disease medium, characterized in that the antigen of the infectious disease medium is increased, the antigen is a first member of a binding pair consisting of a first member and a second member, wherein the method The immunospecific binding level of the modified immunoglobulin to the sample derived from the subject is measured in the subject, wherein the modified immunoglobulin immunospecifically binds to the antigen and the modified immunoglobulin comprises a portion of the second member. Consisting of a variable domain having at least one CDR, with a portion having a binding site to an antigen that does not occur naturally in the CDR; An increase in the level of immunospecific binding as compared to the level of immunospecific binding in a similar sample obtained from an individual without an infectious disease medium indicates that the infectious disease medium is present. A method for treating or preventing cancer in an individual in need of treatment or prevention of cancer characterized by the presence of a cancer antigen, wherein the antigen is a first member of a binding pair consisting of a first member and a second member, and the cancer antigen is modified Immunospecifically binds to immunoglobulins and the modified immunoglobulins consist of a variable domain having at least one CDR comprising a portion of the second member, wherein the portion has a binding site to an antigen that does not occur naturally in the CDRs; The method comprises administering to a subject a therapeutically or prophylactically effective amount of a modified immunoglobulin. In a method for treating or preventing an infectious disease in an individual in need of the treatment or prevention of an infectious disease, characterized by the presence of an infectious disease medium, the antigen is a first member of a binding pair consisting of a first member and a second member, Is immunospecifically bound to the modified immunoglobulin, wherein the modified immunoglobulin consists of a variable domain having at least one CDR comprising a portion of the second member, wherein the portion binds to an antigen that does not occur naturally in the CDR. have; The method comprises administering to a subject a therapeutically or prophylactically effective amount of a modified immunoglobulin. In a method for treating or preventing a disease in an individual in need of treating or preventing a disease by an infectious disease medium that binds to a cell receptor, the cell receptor is a first member of a binding pair consisting of a first member and a second member, and a cell The receptor immunospecifically binds to the modified immunoglobulin, wherein the modified immunoglobulin consists of a variable domain having at least one CDR comprising a portion of the second member, wherein the portion of the binding site is Asn-Ala-Asn-Pro Or a binding moiety to a cellular receptor that does not have a sequence of Asn-Val-Asp-Pro and is not found in nature in the CDRs, wherein the method produces a therapeutic or prophylactically effective amount of a modified immunoglobulin. Administering to the method. A method of modulating the activity of a first member of a binding pair, wherein the binding pair consists of a first and a second member, the method comprising administering a modified immunoglobulin according to claim 1, 13, 23 or 29. How to. In a method of making a modified immunoglobulin that immunospecifically binds to a cancer antigen, the cancer antigen is a first member of a binding pair consisting of a first member and a second member, and the modified immunoglobulin comprises a portion of the second member. Consisting of a variable domain having at least one CDR, with a portion having a binding site to a cancer antigen that does not occur naturally in the CDR; The method of production comprises the steps of growing a recombinant cell comprising a nucleic acid consisting of a nucleotide sequence encoding a modified immunoglobulin such that the cell expresses the encoded modified immunoglobulin and recovering the expressed modified immunoglobulin. Method for producing a modified immunoglobulin, characterized in that. In a method of making a modified immunoglobulin that immunospecifically binds to an antigen of an infectious disease medium, the antigen is a first member of a binding pair consisting of a first member and a second member, and the modified immunoglobulin is a portion of the second member. Consisting of a variable domain having at least one CDR comprising, wherein a portion has a binding site to an antigen that does not occur naturally in the CDR; The method of production comprises the steps of growing a recombinant cell comprising a nucleic acid consisting of a nucleotide sequence encoding a modified immunoglobulin such that the cell expresses the encoded modified immunoglobulin and recovering the expressed modified immunoglobulin. Method for producing a modified immunoglobulin, characterized in that. In a method of making a modified immunoglobulin that immunospecifically binds to a cellular receptor of an infectious disease medium, the cell receptor is a first member of a binding pair consisting of a first member and a second member, and the modified immunoglobulin is a member of the second member. Consisting of a variable domain having at least one CDR comprising a portion, wherein the portion comprises a binding site of a cell receptor that does not occur naturally in the CDR, but not in an Asn-Ala-Asn-Pro or Asn-Val-Asp-Pro sequence Including; The method of production comprises the steps of growing a recombinant cell comprising a nucleic acid consisting of a nucleic acid sequence encoding a modified immunoglobulin such that the cell expresses the encoded modified immunoglobulin and recovering the expressed modified immunoglobulin. Method for producing a modified immunoglobulin, characterized in that. A method of making a modified immunoglobulin that immunospecifically binds to a ligand, wherein the ligand is a first member of a binding pair consisting of a first member and a second member, wherein the modified immunoglobulin comprises at least one portion of a second member Consisting of variable domains having three CDRs, with a portion having a binding site for a ligand that does not occur naturally in the CDRs; The method of production comprises the steps of growing a recombinant cell comprising a nucleic acid consisting of a nucleotide sequence encoding a modified immunoglobulin such that the cell expresses the encoded modified immunoglobulin and recovering the expressed modified immunoglobulin. Method for producing a modified immunoglobulin, characterized in that. A method of making a modified immunoglobulin that immunospecifically binds to a receptor, wherein the receptor is a first member of a binding pair consisting of a first member and a second member, wherein the modified immunoglobulin comprises at least one portion of a second member Consisting of variable domains having three CDRs, some of which have binding sites for receptors that do not occur naturally in the CDRs; The method of production comprises the steps of growing a recombinant cell comprising a nucleic acid consisting of a nucleotide sequence encoding a modified immunoglobulin such that the cell expresses the encoded modified immunoglobulin and recovering the expressed modified immunoglobulin. Method for producing a modified immunoglobulin, characterized in that. A method of making a nucleic acid encoding a modified immunoglobulin according to claim 1, 15, 23, or 29, (a) synthesizing a set of oligonucleotides; Wherein the set consists of an oligonucleotide comprising a portion of the nucleotide sequence encoding a modified immunoglobulin and an oligonucleotide comprising a portion of the nucleotide sequence complementary to the nucleotide sequence encoding a modified immunoglobulin, provided that the modified immunoglobulin is Each oligonucleotide has a terminal sequence that overlaps with another oligonucleotide in the set, except for oligonucleotides comprising nucleotide sequences encoding the N-terminal and C-terminal portions of (b) allow oligonucleotides to hybridize with each other; And (c) ligating the hybridized oligonucleotide so that a nucleic acid comprising a nucleotide sequence encoding a modified immunoglobulin is produced. A method of producing a modified immunoglobulin that immunospecifically binds to a first member of a binding pair, wherein the binding pair consists of a first member and a second member, and the immunoglobulin comprises at least one portion of the second member. Consisting of variable domains having CDRs, with a portion having a binding site for a first member that does not occur naturally in the CDRs; The first member is a cancer antigen; At this time, (a) growing a recombinant cell comprising a nucleic acid produced by the method according to claim 115 such that the cell expresses the encoded modified nucleotides; And (b) recovering the expressed modified immunoglobulin. A method of producing a modified immunoglobulin that immunospecifically binds to a first member of a binding pair, wherein the binding pair consists of a first member and a second member, and the immunoglobulin comprises at least one portion of the second member. Consisting of variable domains having CDRs, with a portion having a binding site for a first member that does not occur naturally in the CDRs; The first member is an antigen of an infectious disease medium; At this time, (a) growing a recombinant cell comprising a nucleic acid produced by the method according to claim 115 such that the cell expresses the encoded modified nucleotides; And (b) recovering the expressed modified immunoglobulin. A method of producing a modified immunoglobulin that immunospecifically binds to a first member of a binding pair, wherein the binding pair consists of a first member and a second member, and the immunoglobulin comprises at least one portion of the second member. Variable domain with two CDRs, the portion of which is not naturally occurring in the CDRs and which has a binding site for a first member that is not an Asn-Ala-Asn-Pro or Asn-Val-Asp-Pro sequence, Member is a cellular receptor for infectious disease medium; At this time, (a) growing a recombinant cell comprising a nucleic acid produced by the method according to claim 115 such that the cell expresses the encoded modified nucleotides; And (b) recovering the expressed modified immunoglobulin. A method of producing a modified immunoglobulin that immunospecifically binds to a first member of a binding pair, wherein the binding pair consists of a first member and a second member, and the immunoglobulin comprises at least one portion of the second member. Consisting of variable domains having CDRs, with a portion having a binding site for a first member that does not occur naturally in the CDRs; The first member is a ligand; At this time, (a) growing a recombinant cell comprising a nucleic acid produced by the method according to claim 115 such that the cell expresses the encoded modified nucleotides; And (b) recovering the expressed modified immunoglobulin. A method of producing a modified immunoglobulin that immunospecifically binds to a first member of a binding pair, wherein the binding pair consists of a first member and a second member, and the immunoglobulin comprises at least one portion of the second member. Consisting of variable domains having CDRs, with a portion having a binding site for a first member that does not occur naturally in the CDRs; The first member is a receptor; At this time, (a) growing a recombinant cell comprising a nucleic acid produced by the method according to claim 115 such that the cell expresses the encoded modified nucleotides; And (b) recovering the expressed modified immunoglobulin. An isolated nucleic acid produced by the method of claim 115. 126. The isolated nucleic acid of claim 121 wherein the nucleic acid is a vector.