JPH06501152A - Novel antibodies for the treatment and prevention of infections in animals and humans - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Background of the invention of novel antibodies for the treatment and prevention of infections in animals and humans Effective drugs for the prevention and treatment of infections in animals and humans have long been needed. has been sought after. Typical methods inhibit the growth of microorganisms and eradicate infectious agents It consists of administering chemical agents that provide the immune system. natural and synthetic chemicals is particularly effective as a treatment for bacterial infections, but there is evidence of the emergence of resistant strains. It is clear that this is a problem. Chemical agents have limited effectiveness in case of viral infection disease severity is usually correlated with immune system status.

For many years, the effectiveness of serum from immune individuals for the prevention and treatment of infectious diseases has been known. It is. However, antibodies in human immune serum involved in effective treatment, i.e. The neutralizing antibody component represents only a very small fraction of total serum antibodies. moreover The use of immune serum may result in a shortage of immune donors due to low levels of neutralizing antibodies. However, due to the cost of treatment, it has recently been limited by the risk of further spread.

The development of monoclonal antibody technology has enabled the production of large quantities of pure antibodies from cell lines free of pathogenic microorganisms. We have provided a means to develop and produce sophisticated mouse monoclonal antibodies. this Using techniques to obtain monoclonal antibodies that interact with pathogenic microorganisms Some of these monoclonal antibodies have been tested in infected mice. was able to prevent the growth of microorganisms. Unfortunately, in vitro studies Research shows which antibodies are most effective at inactivating microorganisms in vivo. It is impossible to predict what will happen. against its target under in vitro set conditions. Many monoclonal antibodies with high binding affinity are effective in vivo. There isn't. In fact, antibodies are effective in protecting against microbial growth under laboratory conditions. (In some cases, these antibodies may not be effective under in vivo conditions.) It has been proven that.

The impact and limitations of mouse monoclonal antibodies on the treatment of infectious diseases show that respiratory Illustrated by the case of syncytial virus (RSV) infection. RSV is an infant is the main cause of lower respiratory tract infection in young bullfinch, and is a significant cause of respiratory disease in young bullfinch. This is the cause. In humans, many attempts to immunize against R8V infection have failed. R with a chemical agent such as ribavirin. Treatment of 3V infection is only partially effective. Mouse mono specific to RSV Clonal antibodies are effective in preventing and treating RSV in mice It is shown. However, for the treatment and prevention of RSV in non-mouse species The use of mouse monoclonal antibodies in Potentially limited by the species' immune response, i.e. human response to mouse antibodies. The human immune response is based on immunoglobulin constant and variable regions (human anti-mouse antibodies). shown in both. Thus, for effective prevention and treatment of R5V infection, Immunoglobulin constant and variable regions are expressed as “self” by R5V-infected recipients. f) Non-immunogen of monoclonal antibodies containing the amino acid sequence recognized as J Sex mutants are required.

Recombinant DNA techniques separate specific immunoglobulin (Ig) regions from one species. Patent Cooperation Treaty that characterizes the ability to modify antibodies to replace regions from species of Neuberger patent application number PCT/GB85100392 er) et al., and Celltech Limited. )) is a method for producing 12 complementary and light chain variable molecules from a species of The domain can be combined with a complementary type and light chain 1g constant domain of another species. It says it can be done. Using this method, e.g. Mouse monoclonals can be modified. Such modifications can be applied to mouse antibodies. by replacing the constant region domain with a human 1piG constant region domain, Create "chimeric" antibodies that could potentially be used to treat such human diseases. death However, such chimeric antibodies are directed against murine (i.e., “exogenous”) variable regions. would still potentially elicit an immune response in humans who do so.

British Patent Application Publication No. GB2188638A (Finter) ) is a method for transferring its complementarity determining regions (CDRs) from a species to another species. describes a method for modifying antibodies by substituting complementarity determining regions from Ru. Using this method, for example, C A DR can be replaced with another CDR from a mouse variable region domain. this Their modified Ig variable region was then combined with the human 1g constant region and replaced. An antibody is created that is entirely human in composition, except for the mouse CDRs. child Mouse CDR-substituted antibodies such as It is likely to induce a significantly reduced immune response in humans compared to chimeric antibodies.

However, as stated in British Patent Application Publication No. GB2188638A, , simply complementing one or more CDRs from another species that is specific for the desired disease. Substituting a complementary CDR with a complementary CDR does not necessarily preserve its antigen-binding ability. It is not always possible to obtain an altered antibody that has . The UK patent application claims that "by routine experimentation or by trial and error" It is proposed that functionally modified antibodies with original binding ability may be obtained. deer and the nature of the routine experimental manipulations or trial-and-error methods required to obtain the desired antibodies. No description is provided, and successive substitutions of CDRs from various sources should be attempted. It has been suggested that it is possible.

Several IgG conformational studies revealed that each of the heavy and light chain 1g variable regions The system consists of three loops supported on a sheet-like structure called a variable area framework. The conclusion was reached that it consists of a group structure (including CDRs). What constitutes a CDR? The rough definition of what constitutes the framework and framework is based on the amino acid sequences of many Ig. It's on.

In a three-dimensional arrangement, the rope structure and CDRs between mouse and human antibodies are There is considerable overlap, but not an exact match. Thus, in some cases, C Transfer of antigen binding specificity by substitution of DH adjacent to that particular CDR This appears to require further substitution of residues. For example, in some cases, variable The framework amino acid residues of the region bind to the antigen through direct interaction with the CDRs. It has been hypothesized that it may be important in binding (Amit ( Amit et al., Science, 233 (1986), 747 ~753 pages.

Queen et al., Proceedings Off National Academy ・Off Science (Proc, Natl^cad, Sci, ), Dan 6 (1989) pp. 10029-10033: and Protein Design La. Protein Design Labs.

Patent Cooperation Treaty patent application publication number WO900 published on July 26, 1990 7861). In the literature cited by Queen et al., they has the greatest correlation to the mouse variable region containing the CDR used for replacement. On this basis, human variable regions for mouse CDR-1 replacement were selected. In addition, Based on computer modeling, Queen et al. CDRs containing several mouse framework amino acids thought to act on A human framework was used for replacement. The resulting modified antibody retains antigen-binding ability but contained additional mouse framework amino acids. This kind of attachment Additive mouse framework amino acids improve immune response to engineered antibodies in humans may contribute to the strengthening of

In addition, previous studies (e.g. Riecbmann et al., Nature (Nature), 332 (1988), pp. 323-327) is a new form. High-affinity binding in vitro was demonstrated in mice using reshap-ing. It has been demonstrated that it can be used to transfer human antibodies from human antibodies, but in Effective protection against human respiratory syncytial virus (RSV) proliferation in vivo It has previously been shown that it is possible to obtain the combination of properties necessary to maintain It has not been.

Thus, engineered antibodies with minimal immunogenicity for preventing and treating infectious diseases There are demands regarding. In addition, radical modification of the variable region framework and It is possible to produce such modified antibodies without their concomitant effects on immunogenicity and immunogenicity. There is a demand for a limiting method. The present invention provides improvements for the prevention and treatment of infectious diseases. by introducing only mutant antibodies and key variable region framework modifications. The present invention provides a method for producing these.

Paramyxoviridae family, RSV, which belongs to the genus Pneumovirus, , is the leading cause of lower respiratory tract infections in young children. -Next infection leads to incomplete immunity infection and reinfection is frequently observed among children. The role of the immune system in human diseases is unclear. It has not been made clear. Developing an effective vaccine using attenuated or killed R3V Previous attempts to protect children have failed, meaning that not only are children not protected; , subsequent infection with RSV sometimes leads to more severe disease than in non-immunized controls. brought about. R3Vg staining is also a major cause of respiratory air infections in young cattle.

Recently, certain immunological and functional properties regarding the antigenic and functional properties of RSV proteins have been published. and molecular information was obtained. RSV fusion protein (F) and R3V attachment protein (G) has been identified as the main viral antigen, and its gene has been cloned. and arranged. Two antigenically distinct cells, designated A and B, A large group of human RSV have been described. Antigens between subgroups A and B The differences are primarily in the RSV G protein. In contrast, RSV F Proteins are divided into two subgroups and various types within these subgroups. There is a high degree of genetic and antigenic homology between the strains.

Monochrome directed against both envelope glycoproteins (F and G) of RSV mAbs were shown to neutralize the virus (Walsh & Walsh & Hruska Journal of Biology (J, Virology).

Dan, 171-177 (1,983); and Warunyu et al. Journal of General Biology (J, Genvirology) , 65. 761-767 (1984)). However, F protein In vitro and in vitro studies with current mAbs or vaccinia virus recombinants. In vivo studies have shown that this protein is most important in inducing cross-protective immunity. (Johnson et al., Journal ・Off Pyrology (J, Virology).

Seeding Off National Academy Off Science Off The. United Staple Off America (Proc, Nat, ^cad, Sci, USA), 7462-7466 (1986) 1 Wertz ( Wertz et al., Journal of Pyrology (J, l'irology) , 61. 293-310 (1987); and Valsh Infection and Immunity immunity), 43. 756-758 (1984)). several authors C. Identify different antigenic sites in the F protein, and identify at least three antigenic sites. has been shown to be involved in neutralization. Two or three different neutralizing epitopes It was confirmed that the protein was located on the F protein using the method described above. escape Using a different virus, Lopez et al. G (JJirologY).

64.927-930 (1990), the F subunit of F protein The two amino acid residues (i.e., 262-Asn and 268-Asn) of shown to be essential for the integrity of individual neutralizing epitopes. kept at a different altitude Neutralizing epitopes have been identified in the journal paper of Trudel et al. General Virology (J, General Virology) 8.2273-2280 (1987), the F protein was synthesized using synthetic peptides. The protein is mapped to residues 221-ile to 232-Glu of the F1 subunit. Ta. Finally, recent analysis using the Pepscan procedure has shown that F-protein Epitope at position 483-Phe to 488-Phe of F1 subunit of proteinaceous has identified an epitope that can be matched to another neutralizing epitope. (Scopes et al., Journal General Pyron) J, General Virology), 71.53-59 (1990) Sansho).

There is a need to develop new therapies for the treatment and prevention of R3V infections. be. Neutralizing and protective epitopes of the R3V viral antigen may help prevent RSV infection and be useful for producing monoclonal antibodies useful in therapy and/or therapy. It was revealed. The present invention provides R5 that is recognized by neutralizing and protective antibodies in vivo. Such novel epitopes on the VF protein are provided.

Figure 1 shows the DNA sequence and corresponding amino acid sequence of the R3V19 heavyweight variable region (VH). Indicates a column. Circle the CDR sequence. Underlined first 8 and last The 11 amino acids correspond to the sequence of the oligonucleotide primer used.

Figure 2 shows the DNA sequence and corresponding amino acid sequence of the RSV19 light chain variable region (VK). Indicates a column. Circle the CDR sequence. Underlined first 8 and last The six amino acids correspond to the sequence of the oligonucleotide primer used.

Figure 3 shows human 1g heavy chain variable region framework and CDRs derived from mouse R3V19. The basic plasmid pHuR5V19VH is shown.

Figure 4 shows human 1g light chain variable region framework and CDRs derived from mouse R3V19. The basic plasmid pHuRsV19VK is shown.

Figure 5 shows RSV19VHSR3V19VK, pHuR5VVH and pHuR8V 19VK and an inducible rg-encoded protein encoded by a derivative of pHuR5V19VH. The variable region amino acid sequence is shown.

Figure 6 shows the HuR3V19VH/VK antibody and its derivative, HuR5V19VHF. ELISA analysis of NS/VK binding to R3V antigen is shown.

Figure 6A shows R3V19 and HuR5V19VHFNS/HuRSV19VK antibodies. Even if there is a difference between the ability to bind to intact non-denatured respiratory syncytial virus, It shows very little.

Figure 7 shows that mAb R3V19 has amino acid residues 417 to 432 of F protein, respectively. and two synthetic peptides consisting of 422-438.

Summary of the invention The present invention provides light and/or heavy variable domains of acceptor monoclonal antibodies. at least some portion of the complementarity determining regions (CDRs) in the replaced by similar portions of the CDRs from the donor monoclonal antibody; To preserve the binding specificity of the donor monoclonal antibody, the acceptor moiety The light and/or heavy variable domain framework regions of noclonal antibodies are minimal. Such donor antibodies, which may be modified or unmodified, are Engineered antibodies (a) with specificity for respiratory syncytial virus (RSV) tered antibody). The present invention also provides such modified antibodies. Therapeutically, a non-toxic amount of such antibody and a pharmaceutically acceptable carrier or diluent. and an effective amount of such engineered antibody for prophylactic or therapeutic use. Is it administered to humans or animals who need to treat microbial-induced disease symptoms? preventive or therapeutic treatment of the disease symptoms in such humans or animals. Regarding how to process. Preferably, the modified antibodies of the invention are produced using recombinant DNA techniques. produced by. The modified antibody of the present invention is a complete antibody (having full length and light chain). molecule or Fab or any thereof such as (F a b':h fragment) Fv or 5CA (single chain antibody) fragment, light chain or heavy dimer or Fv ) or any of the minimal recombinant fragments thereof, such as It may also consist of any other molecule with the same specificity. In addition, modifications of the present invention The antibody may have an effector or reporter molecule attached to it. example For example, a modified antibody of the invention may have a heavy metal atom attached to it by a covalently bridged structure. or may have a large ring to chelate toxins such as ricin . Alternatively, Fc fragments of complete antibody molecules can be engineered using recombinant DNA techniques. of the present invention, in which the target or CH3 domain is replaced by an enzyme or a toxin molecule. Bright modified antibodies may also be produced. The modified antibody reminder may be any suitable It may also be derived from human immunoglobulin. However, human immunoglobulin protein It does not have to consist only of solid arrays. For example, parts of human immunoglobulin chains A DNA sequence encoding a polypeptide effector or reporter molecule It is possible to construct a gene that fuses an amino acid sequence to a DNA sequence encoding it. Ru.

Another aspect of the invention is R3Vl:! I! A module to protect and treat infected mice RSV F (fusion) protein shown to be a target for noclonal antibodies The quality lies in the knowledge of specific epitopes. In addition, such monoclonal antibodies The F a, b fragment protects mice from infection in vivo. It is also shown as (Therefore, the present invention also provides this method of RSV F protein. specific epitopes such as; monoclonal antibodies directed against such epitopes; and Fab fragments of such monoclonal antibodies. In addition, the main Therapeutically, non-toxic amounts of such monoclonal antibodies or Fab Flags and a pharmaceutically acceptable carrier or diluent; Effective doses of such monoclonal antibodies or Fab fragments were administered to R3V infection. from administration to humans or animals in need of prophylactic or therapeutic treatment. to treat R3V infection in humans or animals prophylactically or therapeutically. Regarding how to

The present invention provides modified antibodies having specificity for microorganisms and encoding such antibodies. It provides DNA. These antibodies have a 1g constant region and a and one or more CDRs from different sources.

In addition, amino acid substitutions in the variable region framework can improve antigen binding affinity. It is stated that it is important for The present invention also provides modified antibodies in mammalian cell hosts. The present invention provides vectors for producing the cells.

In particular, the present invention provides properties necessary for the prevention and treatment of infections in animals and humans. It is applied to provide modified antibodies in combination. For example, humanized non-human antibodies that elicit a minimal immune response in humans [humanized (h umanised) J antibodies can be produced. In particular, the present invention provides Effective in animal studies on RSV infection and clinical isolation of diverse human RSV Provides a “humanized” antibody with specificity for RSV that is known to recognize It is something to do.

The present invention also provides variable region Provides a method for making minimal modifications to framework amino acids. The method involves antigen binding Stepwise modifications and changes in the variable region framework potentially important for affinity and individual amino acids. The method uses framework amino acids from the same source as the CDRs. Avoid major introduction of noic acids.

As used herein, the term "humanized antibody" refers to immunoglobulins from non-human species. Its complementarity-determining region derived from phosphorus (and possibly its light and/or heavy A molecule that has a variable domain (the smallest part of the framework region), and the rest of the molecule The immunoglobulin-derived moiety is derived from human immunoglobulin.

The present invention provides light and/or heavy variable domains of acceptor monoclonal antibodies. at least a portion of the complementarity determining regions (CDRs) in the replaced by a similar portion of CDH from the donor monoclonal antibody above. In order to preserve the binding specificity of the donor monoclonal antibody, the acceptor light and/or heavy variable domain framework regions of monoclonal antibodies A modified antibody that may or may not be minimally modified in the region, Such donor antibodies are specific for microorganisms, especially respiratory syncytial virus (RSV). ).Related to a modified antibody having specificity for The present invention also provides for the production of such modified antibodies. Therapeutically, a non-toxic amount of such engineered antibody and a pharmaceutically acceptable carrier or diluent a pharmaceutical composition comprising: and an effective amount of such engineered antibody for treatment of microorganism-induced disease symptoms. administration in humans or animals. It relates to a method of prophylactically or therapeutically treating disease symptoms.

The modified antibodies of the present invention can be produced by (a) conventional techniques. Therefore, an expression vector containing an antibody heavy or light chain-encoding DNA sequence can be used. produce a peron, in which at least the CDRs of the variable domain (and the donor The acceptor moiety required to maintain the binding specificity of monoclonal antibodies that of the light and/or heavy variable domain framework regions of a noclonal antibody; (the smallest portion of the The remaining immunoglobulin-derived portion of the antibody chain is derived from human immunoglobulin. (b) by conventional techniques, thereby producing a vector of the invention; In the expression vector, a DNA sequence encoding a light or heavy complementary antibody is inserted. producing an operon having at least the variable domain CDRs (and Acceptance requirements required to preserve binding specificity of donor monoclonal antibodies the light and/or heavy variable domains of monoclonal antibodies. ) is derived from non-human immunoglobulin, as produced by RSV19. The remaining immunoglobulin-derived portion of the antibody chain is derived from human immunoglobulin. (C) vectors of the invention by conventional techniques; The principal cells are transfected with the or each vector, and the transfection of the present invention is carried out. (d) transfection by conventional techniques; The cells thus obtained are cultured to produce the modified antibody of the present invention.

Host cells can be transfected with two types of vectors, where One vector contains an operon encoding a light chain-derived polypeptide; The vector contains an operon encoding a heavy-duty derived polypeptide. as much as possible The coding sequence and selectable Preferably, the vectors are the same except to the extent that the markers are related. alternatively A single vector of the invention may be used, which carries both light chain and heavy chain derivatives. It includes sequences that together encode a polypeptide. In coding sequences for light and heavy chains The DNA may consist of cDNA, genomic DNA, or both.

The host cells used to express the modified antibodies of the invention are preferably eukaryotic cells, most preferably eukaryotic cells. Also preferably mammalian cells, such as CHO cells or bone marrow cells.

General methods by which vectors of the invention may be constructed, producing host cells of the invention The transfection method required for producing the modified antibodies of the present invention from such host cells. The culture methods required for production are all conventional techniques. Similarly, after production, sulfur Amp precipitation, affinity column, column chromatography, gel electrophoresis, etc. The modified antibodies of the invention are purified by standard techniques in the art, including Good too.

Examples of the modified antibodies of the present invention include HuRSV19VH/VK and Mouse monoclonal like HuR8V19VHFNS/HuR8V19VK This is a Daika antibody derived from antibody R3V19. Such antibodies may be used therapeutically or prophylactically, Useful in human treatment for human RSV infection. Therefore, the present invention also provides It is particularly important to administer an effective amount (a therapeutic dose for human RSV infection) of such engineered antibodies to humans. Therapeutic treatment of human R5V infection in humans requiring treatment of human R5V infection with symptoms or regarding methods of prophylactic treatment.

Additionally, the modified antibody of the present invention may be directed against other antibodies, particularly against which the modified antibody of the present invention is directed. human monoclonal antibodies that are reactive with other markers (epitopes) associated with the disease. May be used in combination with the body.

Furthermore, the modified antibodies of the present invention may be administered in combination with chemotherapeutic agents or immunosuppressants. It may also be used as a separate administration composition. Appropriate combinations of agents to be used can be determined by those skilled in the art. If so, it can be easily determined using conventional techniques. One of such combinations For example, to facilitate the treatment of R3V infection in humans, HuR8V19 The modified antibody of the present invention, known as VHFNS/HuR3V19VK, is It may also be administered in combination with the antiviral drug ribavirin.

One pharmaceutical composition of the invention is an immunotoxin (i.e., characterized by two components). (molecules that are particularly useful for killing selected cells in vitro or in vivo). The invention consists of using the antibodies of the invention to When one component is deposited or absorbed It is a cytotoxic agent that is usually lethal to cells. The second component is the delivery vehicle. It is known as a delivery vehicle, for example, for cancer provides a means for delivering toxic agents to individual cell types, such as different cells. child The two components are usually chemically combined by any of a variety of well-known chemical techniques. are doing. For example, if the cytotoxic agent is a protein and the second component is an intact immunoglobulin. If the linkage is a heterofunctional crosslinker, such as carbodiimide, glycan, etc. It may also be based on taraldehyde or the like. Various immunotoxins Various cytotoxic agents are known to be suitable for use in immunotoxins, In particular, radionuclides, chemotherapeutic drugs such as methotrexate, e.g. Including cytotoxic proteins such as posomal inhibitory proteins (e.g., linno) Good too.

The immunotoxin delivery component comprises a human-like immunoglobulin of the present invention. do. Intact immunoglobulins or binding fragments thereof, e.g. Fab 1 is preferably used. Typically, the antibodies in the immunotoxin are human of the human IgM or rgG isotype, but optionally of other mammalian IgM or rgG isotypes. Constant regions may also be used.

The modified antibodies and pharmaceutical compositions of the invention can be administered, for example, subcutaneously, intramuscularly or intravenously. It is particularly useful for parenteral administration. Compositions for parenteral administration are generally acceptable. A solution of the modified antibody of the present invention dissolved in a carrier, preferably an aqueous carrier, or a reaction thereof. Consists of a mixed solution. For example, water, buffered water, 0.4% saline, 0.3% glycine, etc. Various aqueous carriers may be used. These solutions are sterile and generally contain granules. Does not include. These solutions may be sterilized using conventional (known sterilization techniques) stomach. The composition includes, for example, a pH adjuster and W! to physiological conditions such as buffering agents, etc. It may also contain pharmaceutically acceptable adjuvant substances required for accessibility. Such pharmaceutical products The concentration of the modified antibody of the invention in the agent ranges from less than about 0.5% to typically 1% or less. may vary widely from at least about 1% to 15 or 20% by weight, depending on the amount selected. Depending on the administration method, the raw material is selected based on its liquid capacity, viscosity, etc.

Therefore, the pharmaceutical composition of the invention for intramuscular injection can be prepared by adding 1 mL of sterile buffered water or and 50 mg of the modified antibody of the present invention. Similarly, The pharmaceutical composition of the invention for intravenous infusion is prepared by adding 250 ml of sterile Ringer's solution and and 150m] of the modified antibodies of the present invention. Can be administered parenterally Practical methods for preparing functional compositions are well known and apparent to those skilled in the art; For example, Remington Syns Pharmaceutical Sciences (Remingt on’s Pharmaceutical 5science), 15th edition, Mac ack Publishing Company (1!ack Publishing Comp any), Easton, Pennsylvania. has been done.

The modified antibodies of the invention are stored lyophilized and reconstituted in a suitable carrier before use. be able to. This technique has been shown to be effective with common immunoglobulins Freeze-drying and renaturation techniques known in the art can be used.

Depending on the intended result, the pharmaceutical compositions of the invention may be used for prophylactic and/or therapeutic treatment. Can be administered for. In therapeutic applications, patients who already have a disease cure or at least partially reverse the disease and its complications in patients with Administer an amount of the composition sufficient to suppress. In prophylactic applications, the antibodies of the invention or by administering a composition containing the reaction mixture to a patient who does not yet have a disease state. Increases the resistance of people.

Depending on the dosage level and mode selected by the treating physician, single or multiple doses of the pharmaceutical composition may be administered. Several doses can be administered. In either case, the pharmaceutical composition of the present invention The engineered antibody of the invention should be given in sufficient quantity to effectively treat the patient. It is.

In addition, the modified antibodies of the present invention may be peptidic or non-peptidic, useful in similar therapies as antibodies. May be used for the design and synthesis of any of the tide compounds (mimics). should be noted (e.g. Saragovi et al., Science ( Science), 253.792-795 (1991)).

Another aspect of the invention provides a method for protecting and treating mice infected with R3V. R5V F (fusion) protein shown to be a target for clonal antibodies lies in the knowledge of specific epitopes. Furthermore, the F of such a monoclonal antibody It is shown that ab fragments protect mice from infection in vivo. However, Therefore, the present invention also provides such specific epitopes of the F protein of R3V: Monoclonal antibodies directed against pitopes: and such monoclonal antibodies. Regarding Fab fragments of the body. Further, the present invention provides therapeutic, non-toxic amounts of such Monoclonal antibodies or Fab fragments and pharmaceutically acceptable carriers or a pharmaceutical composition comprising a diluent, and an effective amount of such monoclonal antibody or R3Vg staining, characterized in that the Fab fragment is administered to humans or animals. prophylactically or therapeutically eliminate R8V infection in humans or animals requiring treatment of The invention relates to modified antibodies having specificity for microorganisms and DNA encoding such antibodies. Provide A. These antibodies contain Ig constant and variable regions from one source, and - or more CDRs from different sources.

Furthermore, amino acids in the variable region framework that are important for antigen binding affinity Describe acid substitution. The present invention also provides methods for producing engineered antibodies in mammalian cell hosts. Provide vectors for The present invention relates to the prevention and treatment of infections in animals and humans. It is particularly suitable for providing engineered antibodies with a combination of therapeutic properties. For example, microorganisms engineer a non-human antibody with specificity to induce a minimal immune response in humans Antibodies may also be produced. In particular, the present invention provides protection against R3V infection in humans. effective in animal models, and highly variable in clinical isolates of human R3V. Provide "humanized" antibodies with specificity for R3V that have been shown to recognize .

The present invention also provides variable region fencing in order to retain the antigen binding ability of CDRs from different sources. Provides a method for minimally modifying framework amino acids. The method includes antigen binding Stepwise modifications and changes in the variable region framework potentially important for affinity and testing of individual amino acids. The method uses frameworks from similar sources as CDRs. - Avoid major introduction of amino acids.

EXAMPLES Hereinafter, the present invention will be illustrated by examples, but the present invention is not limited thereto.

Example In the following examples, all necessary restriction enzymes, plasmids, and other reagents are included. and materials were obtained from commercial sources unless otherwise noted.

In the following examples, all universal cloning, ligation and Biike's recombinant DNA method is based in Cold Spring Harbor, New York. Cold Spring Harbor Laboratory published by [Molecular Closing Laboratory] Clonin, A Laboratory Manual (Molecular Clonin) g, A Laboratory manual) J (1982) T. Mania. Edited by T. Maniatis et al. (hereinafter referred to as "Maniatis et al.") Performed as described.

In the following examples, the following abbreviations may be used: dCTP Deoxythi Gin triphosphate dATP deoxyadenosine triphosphate dGTP deoxyguanosine triphosphate dTTP deoxythysiodine t riphosphate) DTT dithiothreitol DMEM Dulbecco modified Eagle medium PBST Tveen 2 Phosphate buffered saline containing 0 (pH 7,5) modified antibody Examples 1-3 describe the production of modified antibodies of the invention.

Example 1 - Production of modified antibodies specific for RSV The source of donor CDRs used to It was a typical murine monoclonal antibody R5V19.

The R3V19 hybridoma cell line was obtained from R.G.16.0 N.N., UK; Burgess, Near Newbury, Institute for Compton Nimal Health, Compton Laboratory (In5titude for A normal Health.

Compton Laboratory), Sierra Aldine ° Tiller (Ger. Aldine Taylor). Things specific to RSV A method for isolating hybridoma cell lines secreting clonal antibodies is described by Tiller (Ta Ylor et al., Immunology, 52 (1984) It is disclosed by pages 137-142.

Cytoplasmic RNA is determined by F. Avaloro et al. (1980), mesoplasmic RNA. Methods in Enzymolog R9V19 hybridoma by the method of y), Vol. 65, pp. 718-749. cDNA was prepared from cell lines using Ig variable region primers as follows: For the heavy chain variable (VH) region, primer VHIFOR was synthesized. (5'TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCCA G3'') for the 1g light chain variable (VK) region; OR (5°GTTAGATCTCCAGCTTGGTCCC3°) was used.

The CDNA synthesis reaction was carried out using a total of 50 mL (D, RNA 20m9.0, 4mM VHIFOR or VKIFOR, each 250mM dATPSdCTP, d GTP and dTTP, 50mM Tris-HCI pH 7.5, 751M KCI, 10zMDTT.

3mM MgC1z and RNase inhibitor (Milton Keynes, United Kingdom) Pharmacia (Pharmacia) Consisted of 27 units. 7 samples It was heated at 0°C for 10 minutes and slowly cooled to 42°C over 30 minutes. Then, 100mM MLV Reverse Transcriptase (Library, Paisley, United Kingdom) Add LifeT technologies and incubate at 42°C. Incubation continued for 1 hour.

Then, Saiki et al., Science, 239, ( 1988), pp. 487-491. VH and VK cDNAs were amplified. Regarding this type of PCR, the protocol used is The timer is the following VHIBACK (5'AGGTSMARCTGCAGSAGTC WGG3°), and VKIBACK(5°GACATTCAGCTGACCCAGTCTCCA3' ) (where M=C or A, S=C or G, W=A or T).

Primers VHIFOR, VKIFOR, VHIBACK and VKIBACK l and their use for PCR amplification of mouse IgDNA. (○rlandi) et al., Prondinges Off National Academy ・Off Sciences-, :L--! Sue (Proc, Nat, Ac ad, Sci, USA), 86.3833-3937 (1989). It has been disclosed.

V H (7) P CR [I: Seki Teha, DNA/primer mixture is RNA /cDNA High Print 5 Tomi! and Ryo5 Tomi M VHIFOR and VHIBA It consisted of CK primer. For PCR amplification of VK, DNA/ply The mer mixture contains RNA/cDNA hybrid 5xl and domain 51M VHIF It consisted of OR and VKIBACK primers. Each of these mixtures 20ChM dATP, dCTP, dGTP and dTTP, IOIM Tris (Tris)-HC/(pH8゜3), 5QzMKC1,l, DIM MgCl 2.0.01% (w/v) gelatin, 0.01% (v/v) Tween (Tve) en)　20. 0.01% (v/v) Nonidet P2O and Taq DNA polymerase (Cleveland, Ohio, USA) United Stake Biochemicals (United 5tatres) 2 units of Biochemicals) were added. Sample at 94℃ for 1 minute , 60℃, 1 minute.

It was subjected to 25 thermal cycles of PCR at 72°C for 2 minutes, and finally at 72°C for 5 minutes.

For cloning and sequencing, amplified VHD DNA was stored in low melting point agarose. On gel and E1tip-d column chromatography (Duso, Germany) Schleicher and Nyur (Schleicher and 5) phage N113 (Milton, United Kingdom). It was cloned into Ins Pharmacia (Pharmacia). universal black Co., Ltd., Cold Spring Harbor, New York Cold Spring Harbor Laboratory "Molecular Cloth" published by Bor Laboratory Cloning, A Laboratory Manual (Molecular Clonin g, A. Laboratory manual) J (1982) T. mania. Nice (T, Maniatis) et al. W (hereinafter referred to as "Maniatis et al.") Followed the instructions. VHDNA is Ml 3 mpl 8/19 (Consolidated Kingdom, Mil P M13tg131 (Little Chalfont, United Kingdom) after digestion by stT Amersham I interna tional)) into the Pst1 site. Gel purify the amplified VK in the same way. and cloned by the following alternative method. Pvull digestion to M13mp19 (Sma1 site) M13mp18/19 (Sma1 site) PvulI and BglI [digested M13tg 131 (EcoRV-Bgln site) pvulI and BglI [digested M BglII digestion into 13tg131 (Smal-BgLII site).

The resulting collection of duplicate clones was purified using Sequenase (Ohio, USA). United Stake Biochemicals, Cleveland, State using the dideoxy method (States B iochemicals). Snger et al., Proceedings of the National Academy of Sciences ・Off Sciences Ninisei (Proc, Nat, Acad, S ci, USA), 74 (1977), pp. 5463-5467). Established.

From the sequences of RSV19VH and VK as shown in Figures 1 and 2, respectively. , CDR sequences using computer-assisted alignment with other VH and VK sequences. Sequences Off Proteins Off Immunological Increments Sequences of Proteins of Immunolo (Nice-x Dept Off Health A) Human Services, Nis Government Printing ・Office (US Dept. of Health Anct HumanSe rvices, US Government Printing 0ffic eX1987)) according to the method of Kabat et al. Ta.

Transfer of murine RSV19 CDRs into human framework is site-directed mutagenesis Achieved by induction. The primers used were as follows.

VHCDRI 5’CTGTCTCACCCAGTGCATATAGTAGTC GCTGAAGGTGAAGCCAGACACGGT3'VHCDR25°CA TTGTCACTCTGCCCTGGAACTTCGGGGCATATGGAA CATCATCATTCTCCAGGATCAATCCA3' VHCDR35’CCCTTGGCCCCAGTGGTCAAAGTCACTC CCCCATCTTGCAACAATA3゜VKCDRI　5’CTGCTGGT ACCATTCTAAATAGGTGTTTCCATCAGTATGTACAA GGGTCTGACTAGATCTACAGGTGATGGTCA3'VKCD R25’GCTTGGCACACCAGAAAATCGGTTGGAAAACTC TGTAGATCAGCAG3'VKCDR35'CCCTTGGCCGAAC GTCCGAGGAAGATGTGAACCTTGAAAGCAGTAGTAG The DNA template for GT3' mutagenesis was for the VH and VK domains. , respectively, substitution of amino acid 27 from serine to phenylalanine (Liechmann (Ri Echmann et al., see cited references above). , NEW (Saul et al., Journal of Biological Chemistry) Tory (J. Biol. Chem.), 53 (1978), pp. 585-597. ), and REIC Nip (Epp) et al., European Japan Null off biochemistry (Eur, J.

Biochem, ), 45 (1974,), pp. 513-524. It consisted of human framework regions derived from unrelated to CDR M13-based templates consisting of human frameworks were prepared by Liechman et al., Nature. (Nature), 332 (1988).

Oligonucleotide site-directed mutagenesis of human VH and VK genes is Nakamaye et al., Nucriac Assos Off Research (Nucl.

Based on the method of Ac1ds Res, ) 14 (1986) pp. 9679-9698. there was.

VH or VK-terminal chain in M13 1) 3 mouse CDRs in NA 5as+ (Complementarity determining region) Three VH or VK phosphorylated oligonucleotides encoding the sequence A 2-fold molar excess of each of the leotides was added. Primer by heating to 70℃ was annealed in the mold and slowly cooled to 37°C. Flenow fragment (Life Technolon, Paisley, United Kingdom) with a reaction volume of 5 Qml. )6u:T4 CNA ligase (Life Technology, Paisley, United Kingdom) 6u, 0.5 M each of the following nucleoside triphosphates CdATP, dGT P, dTTP and 2′-thioxycytidine 5′-0-(1-thiotriphosphate (thiodCTP) + 60 M tris-HC1 (pH 8,0): 6 gM MgCIx: 5mM DTT (Sigma, Poole, United Kingdom) ): and 10mM ATP were added. This mixture was inked at 16°C for 15 hours. It was incubated. The DNA is then ethanol precipitated, cutting the parent strand but leaving the intact chain. Nci I (Paise, United Kingdom) leaving a newly synthesized chain containing odCTP Lee's Life Technologies) Digested in 5 units. Then 60 of 5Qxl Wings M Tris-HCI (pH 8,0), 0.66 MgCl2, and 11M Exonuclease in DTT (Pharma Pharma, Milton Keynes, United Kingdom) The parent strand was removed by digestion with 100 units for 30 minutes. then , 5Qxl of 6 (0 ton M Tris~HCI! (pH 8,0), 6 dew M Mg (J’ ,, 5 proverbs M DTT, 1011M ATP and 05 Otori M dATP, dC respectively DNA polymerase I in TP, dGTP and dTTP (Paisley, United Kingdom) - Addition of 3 units of T4 DNA ligase (Life Technologies) and 2 units of T4 DNA ligase Repaired via. The DNA was transformed into competent cells by the method of Maniais et al. - E. coli TG 1 cell (Little Chalfont, United Kingdom, Amarjam International).

Messing's method [(1983), Methods in Enza Immolon-CMethods in Enzymology), 101, No. 20 Single-stranded DNA was prepared from individual plaques and sequenced by . Once single or double mutants are obtained, triple CDR mutants are obtained. Then, these were mutagenized using appropriate oligonucleotides. It was put through the circulation.

Cloning the CDR-replaced VF and VK genes in an expression vector (manipulator) (according to the method of Anais et al.), the plasmids shown in Figures 3 and 4 were obtained, respectively. . The Bluff and MiF are referred to as pHuR3V19VH and pHuR5V19VK. did.

For pHuR3V19VH, the Ig heavy chain promoter (Figures 3 and 4) , appropriate splice site and signal peptide sequences (Figures 3 and 4) - The original CDR-replaced VH gene was digested with HindDI and BamHI. was excised from M13, and the murine 1g heavy-weight enhancer (Figures 3 and 4), SV40 promoter (Figures 3 and 4), for selection in mammalian cells. gpt gene (Figures 3 and 4) and E. coli. Expression vectors containing genes for replication and selection (Figures 3 and 4) cloned into a vector. The human) IgG1 constant region was then converted into a BamHI fragment. (Figures 3 and 4).

The construction of the pHuR3V19VK plasmid was carried out in such a way that the gpt gene was resistant to hygromycin. (Figures 3 and 4), human kappa chain constant They were substantially the same except for the addition of regions (Figures 3 and 4).

pHuR3V19VH10m9 and pHuR5V19V using conventional methods. K20 rice was digested with pvul. DNA was mixed together, ethanol precipitated, and water It was dissolved in 25d. Approximately 10' (YB210 cells from Ill. - American Type Culture Collection, Rockville, Land. ericanTypeCultureCo11ection)) to semi-dense DME along with the digested DNA was grown, harvested by centrifugation, and digested in a cuvette. M (Gibco, Paisley, United Kingdom) Q, resuspended in 5 ml.

After 5 minutes on water, the cells were given a single pulse of 170V at 960uF (American Bio-Rad, Suchmond, Calif. Gene-Pulser) and left in the water for an additional 20 minutes. Ta. Then, the cells were placed in DM E M ÷ 10% / 20% of infant serum, Played for 48 hours. Cells were then distributed into 24-well plates and selective medium was added. (DMEM, 10% bovine fetal dish serum, 0.8 g/t. mycophenolic acid) and 250 mq/mn xanthine). After 3-4 days, remove the medium and dead cells. and replaced with fresh selection medium. 10-12 transfected clones It could be seen with the naked eye after a few days.

Culture of wells containing transfected cells by conventional ELISA method. The presence of human antibodies in the soil was determined. Microtiter plate II+ per well 9 goat anti-human 1. gG (gamma chain specific) antibody (United Kingdom, Crowley Dow) Sera-Lab-Ltd, ) overnight at 4°C. coated. PBST (0.02% Tveen) 20 x (pH 7,5) After washing with phosphate buffered saline containing 10Qml of culture medium from wells containing Added for a while. The wells were then emptied and washed with PBST and peroxidase. - Conjugate Goat anti-human IgG or peroxidase conjugate ・Goat anti-human kappa constant region antibody (both from United Kingdom, Crawley Down, SE) (obtained from Larraboo Limited) was added at 1100 n per well.

The plates were then incubated at 37°C for 1 hour. Then empty the well. and washed with PBST. 5QmM sodium citrate, 5(bM sodium phosphate) 340-q/mt in H202 (pH 5,0) and 0.003% (v/v) ! of O-phenylenediamine was added at 200 iIl per well. Well hit The reaction is stopped after 1-5 minutes by adding 12.5% sulfuric acid at 50@i It was done. The absorbance at 492r+m was then measured spectrophotometrically.

transfected with pHuR8VVH and pHuRsVVK. Humanized antibody HuR5V1 secreted from fect cell line 9VH/VK was coated with a protein A agarose column (Boehringer, Leunis, United Kingdom). Purified on Boehringer Mannheim and ELI Tested for binding to R8V virus in SA assay. Liar Kidney (CK) The antigen consisting of cells is RSV (RSV A2 bacterium obtained from a child in Australia). LeWis et al., Med, J.

Au5tralia, 48.932-933 (1961)). Cell lysates were obtained by staining and treatment with 0.5% (v/v) NP40 detergent. similar Control cell lysates were prepared using uninfected CK cells. Microtitration plate Wells were coated with either infected or control cell lysates. Antigen coating Rates were blocked with PBST for 1 hour at 37°C, washed with PBST, and then , a humanized antibody was applied (ie, HuR5V19VH/VK). 1 at 37℃ After an hour, the wells were emptied, washed with PBST, and goat anti-human IgG antibody was added per well. Body (United Kingdom, Crowley Down, Sarah Love Limited) 20On9 Added. After 1 hour at 37°C, the wells were emptied, washed with PBST, and rinsed with hosela. Ditsch peroxidase, conjugated rabbit anti-goat IgG antibody (linked) 1:1000 dilution solution 2001j' of Nogma in Poole, United Kingdom was added.

After 1 hour at 37°C, the wells were emptied and washed with PBST. Add substrate to each well. Solution (50M sodium citrate, 501M sodium phosphate (pH 5.0) and 340 mg/ml 0-phenylenedia in 1003% (V/V) H202. 12.5% sulfuric acid 5ONl was added. has been stopped. Then, the absorbance at 492 nll was measured. Antibody HuR, 5VV H/VK bound RSV with lower affinity than the murine R319 antibody.

Example 2 - To achieve minimal variable region framework modifications that result in high affinity binding (Production of high affinity antibodies specific for RSV by a designed method) The method of the invention includes the following sequential modification and testing steps: 1. Interaction with CDRs Individual framework amino acid residues known to be critical for action are compared in the primary antibody and the modified CDR replacement antibody.

For example, key amino acid residue 94 (Kabat number, Kabat et al., supra) (see cited references) in donor and modified antibodies. this The arginine residue at position mutually interacts with the invariant heavy CDR aspartic acid residue at position 101. It is thought that it works.

Amino acid 94 is not in the modified antibody framework but in the primary antibody framework. When consisting of arginine, the secondary heavy chain gene consisting of arginine 94 of the modified antibody is produced. will be built. In the opposite case, i.e. the modified antibody framework rather than the -next antibody In the case of an arginine residue at position 4, a sub-heavy chain consisting of the initial amino acid at position 94 Genes are manufactured. Before further analysis, the vice plus produced on this basis Mido will be tested for the production of high affinity engineered antibodies.

2 Four CDRs defined according to Kabat (Kabat et al., supra) The framework amino acids within the residues of are compared in the primary antibody. Where there are differences If, for each region (e.g. upstream of VHCDRI), specific amino acids of that region are substituted with those in the corresponding region of the modified antibody to obtain a small number of modified genes. Based on this The accessory plasmids produced are then tested for production of high affinity antibodies.

3. Compare framework residues in next and modified CDR substitution antibodies and determine charge , highlighting residues with large differences in size or hydrophobicity. to this Based on - the individual highlights represented by the corresponding amino acids of the next antibody. A secondary plasmid is produced with amino acids, and the secondary plasmid is used for the production of high affinity antibodies. Test.

The method uses HuR3VVH/VK specific for RSV (see Example 1). exemplified by the production of high affinity engineered antibodies derived from. RSV19VH A comparison of the VH gene sequences between pHuRsV19VH and pHuRsV19VH (see Figure 5) shows that four Among the amino acid differences, three are between amino acids 27-30 and amino acids 91-94. Show that this occurs. (Shite, pHuR8V19VHNIK and pHuR8 In the former case, V19VHFNS is the frame represented by − next R3V19VH. work amino acids 27-30 and 91-94 and in the latter amino acid 91 and manufactured in 94. Oligonucleotide site-specific targeting described in Example 1 For mutagenesis of the HuR3V19VH gene of M13 using natural mutagenesis. The following oligonucleotides were used to pHuR5V19VHNIK -5' ATATAGTAGTCTTTAATGT TGAAGCCAGACA3' pHuR3V19VHFNs-5'CTCCCCCATGAATTACAGA AATAGACCG3'.

In an ELISA assay as detailed in Example 1, detergent-extracted virus susceptibility Humanized HuR3 for analysis of binding to R5V antigen prepared from stained cells The V19VHFNS/HuR3V19VK antibody was tested. Figure 6 shows HuR5V ■H residues 91-94 in 19VH/vK, V derived from mouse R8V19VH It is shown that substitution with H residue partially restored the antigen binding level. None as an antigen Hu using ELISA assay using type A respiratory syncytial virus (long strain) Further analysis of FNS binding properties was performed. This further analytical ability (shown in Figure 6) are R3M19 and HuR3V19VHFNS/HuR Differences in the ability of the 3V19VK antibody to bind to intact non-denatured respiratory syncytial virus Mo/JX Sako Tora is shown. Concerning analysis, R2M17 and 1tHuR3V 19VHFNS/HuR3V λ is much lower than that seen with 19VK, but HuR Detectable binding of 8V19VH/VK to intact virus was also demonstrated. Thus, this Furthermore, the analytical power of HuRS (7), the affinity for natural antigens is suggesting recovery in V19VHFNS/HuR3V19VK mAb. . RSV F protein I versus HuR3V19VHFNS/HuRSV19VK The specificity is that the truncated soluble F protein expressed in CH○ cells (two L\) This was demonstrated by conventional Western Pro-Soto analysis using the construct.

Example 3 - Specificity and biological activity of engineered antibodies specific for RSV, To evaluate the effective clinical utility of humanized antibodies specific for SV, , immunofluorescence analysis of binding to 24RSV clinical isolates was performed. The isolate ('1 Bristol Pub lic HealthL laboratory) (Bristol, UK) Therefore, during the winter of 1983-84 (collected from two children, RSV It represents both major subgroups. 13 isolates as subgroup A, 1 One isolate was serotyped as subgroup B. Infected with R3V isolates HeLa or MA104 cells were grown in tissue culture. Cells become cytopathic 0.02% (w/v) EDTA (ethylene chloride) in PBS showed evidence of efficacy. aminetetraacetic acid) 201Il and 0.25% (w/v) in PBS Add 3xll of trypsin and transfer the cell suspension to a PTFE-coated slide (polytetra fluoroethylene coated slides) (He (ndley)). After 3 hours at 37°C, the slides were It was dried and fixed in 80% acetone. Cells were incubated for 1 hour at room temperature for monoclonal antibody (i.e., humanized antibody HuR5V19VHFNS/HuR8~'19~' 1 or murine antibody R3V19). After extensive cleaning, Fluorescein conjugated rabbit anti-mouse IgG (Chilburu, The Netherlands) Nordic Laboratories )) or fluorescein-conjugated goat anti-human IgG1 (American Southern Biotechnology, Birmingham, Alabama n　Biotechnology)) and incubation. repeated. After further washing, cells were placed in glycerol and incubated under UV light. I tried it.

Table 1 lists the humanized antibodies HuRSV19VHFNS/HuR3V19VK and Comparative immunofluorescence results for murine antibody R5V19 are shown. This data is clinical 100% of biological isolates are recognized by both humanized and humanized antibodies Show that. These data indicate that humanized antibodies are effective against both major R3V subgroups. demonstrated efficacy for the recognition of most clinical isolates consisting of .

! 1 sachet Binding of humanized anti-R3V to clinical isolates + represents the relative number of fluorescent cells observed and represents the percentage of infected cells.

We then tested humanized cells for biological activity in vitro in fusion inhibition assays. Antibody HuR3V19VHFNS/HuR8V19VK was tested. MA104 thin The suspension of cells was prepared at 0.0 IPFU (plaque forming unit) per cell. infected with R3V at o, i, (multiplicity of infection). After 1 hour at 37°C, 10 '/mj! Cells 2111 were distributed on glass coverslips in tubes. 3 After another 24 hours at 7°C, the culture medium! 'l) antibody HuR3V19VHFNS/H The medium was replaced with a medium containing a dilution of uR3V19VK. After 24 hours, cover slip Cultures were fixed in methanol for 10 min and prepared by May Gr. unvald) Dye (BdH Chemicals Limited, Poole, United Kingdom) (BDH Chemicals Ltd.). Table 2 C, giant cells Suppression of 171MIf +1: HuR3V19VHFNS/HuR3V19V in The effect of increasing concentrations of K is shown. The data shown in Table 2 below are based on RSV type A induced Cell fusion inhibition Niokel humanized antibody HuR8V19VHFNS/HuRSV19 Biological activity of VK. RSVI9 vs. HuR3V19VHFNS/HuR Fusion inhibition titers for 8V19VK are comparable and The affinity was fully restored in HuRSV19VHFNS/HuRSV19VK. It should be noted that further studies provided further evidence that It is possible.

The humanized antibody HuR8V19VHFNS/HuR3V19VK (type A RSV-induced (using a method similar to that used above to demonstrate inhibition of cell fusion) It was also found that type B R3V (strain 8/60) showed dose-dependent suppression of giant cell fusion. I made it clear.

Emu The number of cells with two or more nuclei was counted at 20@2 with an 825X objective microscope lens.

Next, the humanized antibody HuR5V 9VHFNS/HuR3V19VK was added to the R8V antibody. ) was tested for in vivo product biological activity in an infection model. BAL B/C Mouse (Charles Rivers included) The A2 strain of human RSV (Category 4 standard, which does not have a specific pathogen) was Taylor et al., In7 Ekunyong and Immunity (I) nfection and Immunity), 43 (1984), No. 64 Pages 9 to 655 +: Listed) 10' P F U Te nasal cavity attack did. One day before virus infection or 4 days after infection, mice were given a group anti-inflammatory drug. 25 bodies and 9 wings were administered.

Administration of the antibody can also be by intranasal (i,n,) or intraperitoneal (i,p,) route. It was. Five days after R8V infection, mice were sacrificed and lungs were probed for R5V PFU. Assayed (Tiller et al., Infection and Immunity, 43(1) 984), pp. 649-655). The data in Table 3 below is 2 per mouse. HuRSV19VHFNS/HuR5V19VK is R3V with a single dose of 589 Shows to be highly effective in preventing and treating infections.

K-1 is HuR3V19VHFNS/HuR5V19VK anti-infection before R3V3V infection. +4 represents administration of antibody 4 days after infection.

'i, p, - intraperitoneal, 1. n, - intranasal;

- Viral PFU is 10% (W/V) lung homogenate adjusted to PFU per gram of lung. expressed as virus titers from dilutions of Ginate (Tiller et al., supra). vinegar.

<1.71 og+o PFU/g starting dilution of 10% lung homogenate This means that no virus was detected.

HuR3V19VHFNS/HuRSV19VK uses the same method as above. When administered prophylactically to mice challenged with type B R3V (strain 8/60), It was shown to be active in vivo. Furthermore, humanized antibody HuRSV1 9VHFNS/VK was isolated from RSV type B (strain 8) using a method similar to that described above. Active in vitro when administered prophylactically to mice challenged with It was also shown that

The present invention provides that R3V19 or R3V2O is a donor monoclonal antibody. Administering an effective human R9V infection-inhibiting dose of the modified antibody of the invention to a human in need thereof It also relates to a method for preventing human RSV infection in a human comprising:

The present invention provides that RSV19 or R5V2O is a donor monoclonal antibody. Administering an effective human RsV infection-inhibiting dose of the modified antibody of the invention to a human in need thereof A method of therapeutically treating human R5V infection in a human comprising: Also related.

To effectively prevent R5V infection in humans, R3V19 or R8V2O is HuRSV19VH/VK or HuRSV19VHFNS/HuR3V19 Approximately 119/h modified antibodies of the present invention which are donor monoclonal antibodies such as VK A single dose of 9 to 20 xo/kq is administered parenterally, preferably i, v, (intravenous). intramuscularly) or i,m, (intramuscularly); A single dose of 200μ9/kg to about 2119/kg i, n, (intranasally) should be administered at Preferably, such dosages are of the R8V season. Starting at the beginning (October-November) and ending at the end of the R3V season (March-April) Should be repeated every 6 weeks. Alternatively, at the beginning of the R3V season , HuR3V19VH/VK or HuR5V19VHFNS/HuR5V19 R3V19 or R5V2O like VK is the donor monoclonal antibody A single dose of about 5 wql & 9 to about 100 u/kq of the modified antibody of the present invention can be administered i, v, or or i, rn; or such an antibody. A single dose of 9/kg to 10 u/bg should be administered i, n. human HuR3V19VH/ VK also 1; LHuR3V19VHFNS/HuR8V19VK (71 like R About the modified antibodies of the invention, wherein 8V19 or R20 is the donor monoclonal antibody Parenterally in a single dose of 2-g/kg to about 20 g/kg, preferably i, V, or is i4. or approximately 200 μg/kg of such antibody. ~2 9/by should be administered i, n. Such administration If desired, it can be repeated at appropriate time intervals until the R3V infection is eradicated.

Modified antibodies of the invention can also be administered by inhalation. "Inhalation" means intranasal and Indicates oral inhalation administration. such as aerosol formulations or graduated dose inhalers, Dosage forms suitable for such administration may be prepared by conventional techniques. for example, Composition for administration by inhalation on an aerosol container with a capacity of 15-20 mA' To prepare the product, a lubricant such as polysorbate 85 or oleic acid 0. 2-0.2% and 10 modified antibodies of the present invention, and the mixture was Preferably (1,2-dichlorotetrafluoroethane) or and difluorochloromethane for intranasal or oral inhalation administration. into a suitable aerosol container. As a further example, a tolerance of 15 For compositions for administration by inhalation, for aerosol containers with ~20 ml Regarding this, dissolve 1011 g of the modified antibody of the present invention in ethanol (6-8*l) and add the port. adding 0.1-0.2% of a lubricant such as Resorbate 85 or oleic acid; Preferably (1,2-dichlorotetrafluor) in a propellant such as Freon. oroethane) and difluorochloromethane, intranasally or is placed in a suitable aerosol container suitable for oral inhalation administration.

Preferably, the required human R8V infection to be administered by inhalation or The daily dose to be used of the modified antibodies of the invention for therapeutic treatment is is about 0.119 to about 10+gtilh9.

Natural R,SV infections have been reported in bulls, goats, sheep, and chimpanzees. It was done. Thus, for example, using the method described above, suitable mouse antibodies can be (bovinized) J, appropriate framework region residue modifications are made, Specific binding affinity can be restored if desired. Appropriate mouse antibodies are generated If so, one of ordinary skill in the art can use conventional dosing techniques to prevent bovine R3V infection. appropriate dosages and regimens necessary for therapeutically or therapeutically effective treatment. can be determined easily.

Examples 1 to 3 show that modified antibodies for prevention and treatment of infection are modified antibody recipes. using a variable region framework that is effectively recognized as “self” by the can be manufactured. A small modification to the variable region framework is regarding the recipe end. due to the large increase in antigen binding without appreciably increasing immunogenicity. It can be done. Such modified antibodies can effectively prevent and eradicate infection.

Thus, the present invention provides a method for determining complementarity determining sites (C) in heavy or light chain variable domains. DR) properties necessary for effective prevention and treatment of infectious diseases in animals or humans. Substitution of similar portions of CDRs from different sources resulting in antibodies having a combination of The present invention provides a modified antibody that can be used as a modified antibody. Preferably, the entire CDR was replaced. Like Alternatively, the variable domains in the heavy and light chains were modified by CDR substitutions.

Typically, CDRs from a mouse antibody are grafted onto the framework regions of a human antibody. It will be done. The modified antibody preferably has the structure of the natural antibody or its framework. do.

Preferred antibodies are those directed against respiratory polymorphism virus (RSV). and is preferably specific for the R5V fusion (F) protein. child Particularly preferred antibodies of the species have the following X-terminal variable domains in their heavy and light chains: It has an amino acid sequence (see the amino acid shorthand table below).

Heavy’QVQLQESGPGLVRPSQTLSLTCTVSGF? TAVYC ΔB(*ttt BJ Pushi WGSDFT) HV/GQGTTVTVSS Amino Acid shorthand table Amino acid 3 letter abbreviation 1 letter symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cystine Cys C Glutamine Gin Q Glutamic acid Glu E Glycine cty G Histidine His H Methionine Met M Phenylalanine Phe F Proline Pro P Serin Ser S Threonine Thr T Does not necessarily affect the specificity of antibodies to the R3V fusion (F) protein Instead, such engineered antibodies can be modified by changing the amino acids in the variable region. It will be understood by those skilled in the art that modifications may be made to the heavy and light chain amino acids. As much as 25% of the amino acids are located in either the variable region framework or CDRs. It is anticipated that other amino acids may be substituted in either or both. like this Engineered antibodies can protect against respiratory syncytial virus (RSV) infection in animals and humans. and treatment.

The present invention also provides a recombinant plasmid containing the coding sequence of the modified antibody of the present invention; Mammals transfected with a recombinant plasmid containing the coding sequence for a modified antibody of and celloid systems. Such vectors are prepared by conventional techniques, Immunoglobulin regions containing variable region frameworks and CDRs from heterologous sources and the DNA sequence encoding the modified antibody in an operable manner. and a suitable promoter linked to. Such vectors are transfected mammalian cells.

Furthermore, the present invention provides modified antibodies with increased binding affinity, comprising the following steps: The modifications necessary to produce a modified antibody within the variable region framework of the modified antibody. Includes methods for making minimal modifications to: (a) Analysis of framework amino acids known to be A work amino acid is replaced by a corresponding amino acid from the same source as the CDR Production and testing of modified antibodies; (b) determination of framework amino acids flanking CDRs; analysis and whether one or more amino acids within the four residues of the CDR Production of modified antibodies in which R is substituted by the corresponding amino acid from the same source and Testing: (C) Analysis of framework residues within engineered antibodies and single antibody The amino acids originate from the same source as the CDRs, differing in charge, size, or hydrophobicity as raw materials. Production and testing of engineered antibodies in which the corresponding amino acids have been substituted.

The following examples relate to novel RSV F protein epitopes of the present invention.

Specific RSV F Protein Epitopes The following examples show that RSV-induced in vivo Two monoclonal antibodies protect against and treat mice from infection, targeting the RSV F protein. A linear epitope within a protein (this linear epitope is a conformational onal) may be part of the epitope), with an essential arginine at position 429 amino acid residues 417-438 of the F protein coding sequence containing residues, and is the immunoprotective portion thereof, e.g., amino acid residues 417-- of the F protein coding sequence. 432 and amino acid residues 422-438 of the F protein coding sequence (these include (including, but not limited to) linear epitopes. This new epitome (herein sometimes referred to as “epitopes 417-438”) among other Japanese epitope screening, protective and therapeutic agents against RSV, and special Therefore, it is a suitable target for monoclonal antibodies directed against this epitope.

With knowledge of this epitope, the person skilled in the art knows that a vaccine against RSV suitable synthetic peptides can be defined. Also, epitope 417-43 8 is also useful in the treatment, therapy and/or prevention of human RSV infection in humans. be.

The present invention provides monoclonal antibodies derived from such novel epitopes. Fab fragments as protective and therapeutic agents against in vivo infection by viruses. It also applies to the use of, in particular in the protection against RSV.

The present invention provides a copy of monoclonal antibodies generated against epitopes 417-438. Recombinant plasmids containing such coding sequences and recombinant plusses containing such coding sequences. and mammalian cell lines transfected with the transfected transfectants. vector like this is prepared by conventional techniques and includes the variable region framework and CDRs. The DNA sequence encoding the immunoglobulin region and the DNA sequence encoding the antibody and a suitable promoter operably linked thereto. A vector like this are transfected into transfected mammalian cells by conventional techniques. will be removed.

Example 4 This example is a murine monoclonal antibody against RSV F protein. , shows the production of monoclonal antibodies that protect and treat mice from infection.

Murine monoclonal antibodies (mAbs) 19 and 20 were produced as follows. .

BALB/C7 Us (Charles Rivers) A2 strain of human (H)RSV (non-infected with specific pathogens) obtained from Isu et al. [1961, Medical Journal of Australia (Med, Reported by J. Au5tralia), Transfer, 932-933] X10'PFU was inoculated intranasally (i, n, ) twice, 3 weeks apart. 4 Months apart, these mice were challenged with 127 strains of bovine (B) RSV (institute Institute for Anima l　Health)　[Compton, -'-A Newba (Isolated at Near Newbury, Berks, UK) of 2×10′ PFU were inoculated intraperitoneally (i,p). 3 days after vaccination, immune test cells into N5-1 myeloma cells (Williams et al., 1977 , Cell.

12, 663). The resulting hybridoma was previously reported. radioimmunoassay and immunofluorescence (Taylor et al., 1999) 84. Immunology, 52.137-142) Therefore, we screened for antibodies against RSV (Kohle r) et al. s) J, pp397-402. I Lefkovit and Be Bellis ( 1, Lefkovitz & B. Perris)! , Academic Press ( twice on soft agar (as reported by Academic Press) The cloned cells obtained were inoculated into BALB/C mice, and the following As previously reported (Taylor et al., 1984. (see Immunology, 137-142), which produces ascites. Ta.

The specificity of mAbs for viral polypeptides was determined as previously reported ( Kennedy et al., 191118. Journal of Ge Neral Virol/-(J, Gen Virol,), 69.3023-2 032), labeled with <SSS>-methionine or (3H)-glucosamine Radioimmunoprecipitation and immunoplotting of RSV-infected cell lysates Takeda et al., 1985. Electrophoresis phoresis).

β, 492-497). used for immunoplotting method The antigen was used in Hep-2 cells (American type) infected with HR3V A2 strain. ・Culture 0 Collection (American Type Culture) Co11ection), Rockville.

Maryland, USA) or primary chestnut kidney (C) infected with 127 strains of BR5V. K) Cells (Institute for Animal Health) e for Animallleath) [Compton Which one was raw N)? Control uninfected Hep-2 cells or CK cells It was used as an antigen.

Immunoglobulin isotypes of mAbs can be determined using a radioimmunodiffusion kit (Serotic). Ser□tec), Kidlington, Oxford by immunodiffusion method using Don Yaia (Oxfordshire, UK). Decided.

The properties of mAbs 19 and 20 are shown in Table A below.

'ELISAi: Cell antigen toshite, HRSV A2 strain (subtype A), HR Using 3V8/60 strain (subtype B) and BRSV 127 strain, log+o Antibody titer expressed as titer.

5FA = percentage of HRSV strain A2 infected cells showing surface fluorescence.

Virus-infected cells (persistently infected) with 1/100 dilution of 3 mAb and rabbit complement Specific chromium release rate (%) from bovine nasal mucosal cells infected with BRSV.

'　l　50% plaque reduction power expressed as Og+o 50% The day before intranasal infection of RSV strain A2 in the lungs of mice given 100 μl of mAb intraperitoneally. Decrease in peak titer.

Immunoprecipitation of radiolabeled R3V (Brunda et al. (1977)) Null Off Im 10 G (J, rmmunol,), 119.193-1 98) According to the method of demonstrated that it recognizes This indicates that RSV-infected cells are non-reducing and Confirmed by Western plot of reduced lysates. Plot shows HRP junction Goat anti-Mau Mau gG (Kp 1. Gaithersburg) rg), Maryland, USA). mAbs 19 and 20 were The 140k F protein dimer and 70k monomer present in the natural F protein antigen 2 = denatured by boiling in mercaptoethanol The 46K Fl fragment present in the antigen was recognized. mAbs 19 and 20 are It was also identified as IgG2a, and its activity against HRSV A2 and 8760 strains The ELISA titer is similar to its ELISA titer against 127 strains of BRSV. It was. This indicates that the epitope recognized by these mAbs is human R5V. and is conserved among strains of bovine RSV. mAB19 and 20 Both neutralized R5V infectivity and increased the effect on RSV-infected MA104 cells. It showed the formation of multinucleated giant cells. In contrast to mAb19, mAb20 was RSV-infected cells were lysed in the presence of cartilage. mAb19 lyses R3V infected cells Notably, viral infection was detected, as mAb19 stained 88% of the cells. This is not because they cannot bind to the surface of cells. mAb19 and complement in viral infection The inability to lyse cells means that antibody- and complement-mediated lysis is inhibited by this antibody. This indicates that it is not important for mediated in vivo protection. mAb19 and The ability of mAbs 19 and 20 to protect against R5V infection was determined by i, p, inoculation of mAbs 19 and 20. 16n infection of mice with approximately 10' PFU of R8V24 24 hours later. Evaluated by. Lungs of untreated mice killed 5 days after infection were It contained an RSV of 5.51 og+oPFIJ. Here, mAb19 or 2 No virus was detected in the lungs of mice given either 0.

Example 5 This example shows resistance to inhibition by mAbs 19 and 20 generated in Example 4. A method for isolating R3V mutants will be described.

A mutant respiratory syncytial virus that is resistant to neutralization by mAbs 19 and 20 is HRSV It was produced as follows using the plaque reduction method using the A2 strain. tissue culture fla A confluent monolayer of CK cells placed in a SC was incubated with HRSV A2 strain at MOIo of 1. Infected. Starting 24 hours after infection and continuing for 3-5 days, the medium was diluted with 10% Fresh medium containing mAb was replaced daily. Where cell mutation effects were observed I collected the virus. The virus thus prepared was mixed with an equal amount of undiluted mAb. Mix with either 19 or 20 or medium alone for 1 hour at room temperature, then add 35 m m multiwell plate (tank (Nunc), cam strap (Kams) trup), Riskilde, Denmark) on a single CK layer. I planted a seed. After incubation for 1 hour at 37°C, the plates were treated with 0.25% A medium containing agarose and 10% mAb or only medium was overlaid. this Cultures were incubated for 7 days at 37°C with 5% COx in air before vital stain color in 0.15M NaCl to visualize virus plaques. 0.3% 3-(4,5-dimethylthiazolyl-2)-2,5-diphenylte Torazolium bromide was added to the overlay.

Removal of putative mutant viruses from plates containing single plaques onto agar plugs diluted in medium and mixed with an equal volume of mAb 19 or 20 as before, 3 It was seeded onto CK monolayers in 5 mm multiwell plates. Putative mutant virus were removed again in plaque form and placed in a tube with a covering piece of calf testis cells. Inoculated. After 4-6 days of incubation, the cover piece was removed and the mAb1 9 and 20 and FITC-labeled rabbit anti-mouse Ig (Nordic Lapos (Norclic Labs, Tilburg, Netherlands) It was stained with As a positive control, cover strips were treated with polyclonal virus against RSV. Antiserum (Institute for Health-Compton) te for Animal Health-Compton), RSV (produced from hyperimmunized red-throated calves), and FITCI! 1 knowledge Nordic Immunol (obtained from Tilburg, The Netherlands).

Mutant respiratory syncytial virus that did not respond to mAb 19 or 20 by immunofluorescence These viruses were used to infect a monolayer of Hep-2 cells, and E. An antigen for LISA was created. Thus, 3-4 days after R3V infection, cells were Scrape onto the medium, centrifuge at 400 g for 5 min, resuspend in distilled water, and add 0.5% ( v/v) Cell lysates were obtained by treatment with NP40 detergent. Control cell lysate was similarly created using uninfected Hep-2 cells. Against mutant viruses The binding of a panel of mAbs to the F protein of RSV was examined by ELISA. . Fill the wells of the microtiter plate with either infected or control cell lysates. or 50 μm at 37° C. overnight with cordon, PBS and 0.05% Tween. Incubate the ink for 1 hour at room temperature with a blocking buffer consisting of 5% normal pig serum in 20 min. and washed 5 times with PBS/TWEEN. Serial dilutions of mAb (3 steps) was added to the wells and the plates were incubated for 1 hour. PBS/Tween After washing 5 times with (Kp 1. Gaithersburg).

Maryland, USA) was added to each well. most After a final wash, the substrate 3.3°, 5.5’-tetramethylbenzidine (TM B) (ICN Immunobiologicals) Bound zygotes were detected using a method of conjugation (1999, Ill., Ill.).

Mutant viruses selected for resistance to mAb19 showed It did not react with either mAb 19 or 20. Similarly, mA b20 Mutant viruses selected for resistance to mAbs 19 and 20 do not react with mAbs 19 and 20. Ta.

All other mAbs tested showed similar efficacy against the parent HPSV strain A2. It reacted with the mutant at the same time. These results are shown in Table B below.

Table B Table B Binding of anti-FmAb to antibody escape mutants of RSV Example 6 This example describes the amino acid sequence within the F protein that binds to a protective monoclonal antibody. Explaining the identification, in order for the protective mab to bind to this amino acid sequence, the alkaline Indicates that ginine 429 is essential.

infected with either the A2 strain of HR3V or each of the mutants described in Example 5. Using poly(A)'' RNA isolated from cells, we determined the sequence of F protein mRNA. Decided. These sequences are based on the previously reported RSV long F protein sequence ( Lopez et al., 19111g, Virus Research Res, ), 10.249-262) and then the terminal Tracking using deoxynucleotidyl transferase (DeBorde et al. , 1986. Analytical Biochemistry (^nalBjochem) , 157.275-282). It was determined by the dideoxy method (described above) using nucleotide primers. mA Three mutants were selected using b19 and three were selected using mAb20. I chose.

All such mutants have a change at nucleotide 1298 when compared to the parental A2 strain. A single base conversion (C to G) is shown. This nucleotide substitution results in F protein The amino acid residue at position 429 is changed from arginine to serine. mAb1 9 reacted with F subunit in Western blot, so antibody binding The site is F, one of the adjacent amino acids in which residue 429 of the subunit plays an essential role. It is thought that it is determined by the following sequence. F protein amino acid residues 417-4 32.422-438. Synthetic pedals corresponding to 417-438 and 421-450 peptide was tested for its ability to react with mAbs 19 and 20 in an ELISA. Beta. mAbs 19 and 20 contain peptides 417-432 (F417), 41 7-438 and 422-438 (F422), but peptide 431 ~450 did not react. onto the wells of a microtiter plate at 37°C. Coat overnight (“dry”) or over wells for 1 hour at room temperature (“wet”). to coated peptides 417-432 and 422-438 (2 μg/well) The binding of mAb19 is shown in FIG. mAb20 gave essentially the same results. It should be noted that

Example 7 This example shows that Fab fragments derived from mAbs 19 and 20 were used in mice infected with RSV. This shows that it is possible to protect and treat.

mAbs 19 and 20 were produced using Protein A Sepharose (Phar macia), Milton Keynes, Unai Ascites can be removed using Ted Kingdom (United Kingdom). Manufactured. Approximately 10 mg of purified mAbs 19 and 20 were added to the immobilized buffer of O, Dm1. Pine (Pierce-Oud-Bei) (Jerland, Netherlands) at 37°C with constant stirring. Incubated for 5 hours and overnight, respectively. The obtained Fab fragment was immobilized with protein. was collected on a Fin A column (Pierce). Purified 1gG and Papa Fragments of the in cleavage were analyzed by 5DS-PAGE under reducing conditions. Purified 1g G is Ig CDNM4 Oyohi 11M1: jt response, 53.000 d and and 23,000 d band. Protein A fraction containing Fab fragments shows a band at approximately 25.0 OOd, and the fraction containing the Fc fragment is the uncleaved Ig Three variants of approximately 28.0 OOd, corresponding to the heavy and light chains of G and the Fc fragment. It showed a band.

Purified IgG and papain-cleaved fragments were added to Hep cells infected with HR5V A2 strain. -2 cells and uninfected Hep-2 cells as antigens, and HRP-goat Anti-mouse Fab (Sigma Chemical al　Co, ), St. Louis, Mississippi, USA) and ) (RP-Ya anti-mouse FC (ICN ImmunoBiologicals) anti-R3V activity was determined by ELT SA using Examined. ELISA shows that mAb 19 and 20 Fab fragments were contaminated with 1 g of uncleaved It was shown that it has not been done. These data are shown in Table C below.

Table C. Prophylactic and therapeutic effects of Fab fragments against R5V infection in mice. Titer was determined by ELISA using R8V/A2 and antigen.

The concentration of antibody in uncleaved mAbs 19 and 20 was Modulate AS titers to protect against RSV infection in BALB/c mice I investigated the ability to Groups of 5 mice received uncleaved purified mA. b19 or mAb20 or Fab fragment (derived from mAb19 or mAb20) before IS inoculation with approximately 10' PFU of HR8V A2 strain Inoculated i, n after 1B. Control mice were inoculated with HR3V only. cormorant Five days after viral infection, mice were killed and their lungs were harvested as previously reported. (Taylor et al., 1984. Infection and Im Infect Immun, ), 43°649-655), secondary C R3V PFU on K cells was assayed. mAbs 19 and 20 Fab fragments are highly effective at protecting against RSV infection and eradicating established infections. It was fruitful.

The present invention relates to epitopes 417-438. Furthermore, the present invention provides epitope 4 Also relates to monoclonal antibodies raised against 17-438. things like this Clonal antibodies are produced by conventional techniques, e.g. including natural antibodies, human monoclonal antibodies, and bovine monoclonal antibodies. However, it is not limited to these. Such monoclonal antibodies are complete antibody molecules. (with full-length heavy and light chains) or fragments thereof, such as Fab or (Fa b') z fragments, light and heavy chain dimers or minimal recombinant fragments thereof, such as Fv and 5 CA (single chain antibodies) or other molecules with the same specificity as monoclonal antibodies Consisting of

The present invention also provides monoclonal antibodies generated against epitopes 417 to 438. The present invention also relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a pharmaceutically acceptable carrier or diluent.

The present invention also provides monoclonal antibodies generated against epitopes 417 to 438. the body required to protect against human R3V infection at a dose effective to inhibit human R3V infection. Prevention of human R3V infection in such humans consists of administering it to humans who require it. It also relates to how to do it.

The present invention also provides monoclonal antibodies generated against epitopes 417 to 438. the body required to treat human R8V infection at a dose effective to treat human R3V infection. The treatment of human RSVg infection in such humans consists of administering it to a human in need. It also relates to a method of treatment.

To effectively protect against R5V infection in humans, The produced monoclonal antibody is approximately 1 mg/kg to approximately 20 mg/k per treatment. Parenterally, preferably i, v, (intravenously) or ]1 m, (intramuscularly) at a dose of ) or administering such antibodies at approximately 200 μg/kg per dose. Should be administered i, n (intranasally) at a dose of ~2 mg/kg. Like Alternatively, such doses should be started at the beginning of the R5V season (October-November). and repeated every six (6) weeks until the end of the RSV season (March-April). There must be.

Alternatively, at the beginning of the R5V season, About 5 mg/kg to about 100 mg/kg of monoclonal antibody In dose: 1. ■, or i, m, or administering such an antibody, 0.5mg/kg to about 10mg/kg per dose 1. n, must be administered do not have.

To effectively treat R3V infection in humans, The produced monoclonal antibody is about 2 mg/kg to about 20 mg/kg per time. is administered parenterally, preferably i, V, or 11 m, at a dose of Alternatively, such antibodies may be administered at a dose of about 200 μg/kg to about 2 mg/kg per dose. The amount must be administered 1n. Such doses, if necessary, Repeat at appropriate time intervals until the infection is eradicated.

In addition, monoclonal antibodies generated against epitopes 417-438 It may also be administered by. "Inhalation" means intranasal and oral inhalation administration do. A suitable dosage form for such administration, e.g. an aerosol formulation or Metered dose inhalers may be prepared by conventional techniques. For example, capacity 1 To prepare compositions for inhalation administration in 5-20 ml aerosol containers, 02 mg of monoclonal antibody generated against pitope 417-438 ~0.2% of a lubricant such as polysorbate 85 or oleic acid; A mixture such as a propellant such as Freon, preferably (1,2-chlorotetate) It is dispersed in a combination of fluorocoethane) and difluorochloromethane and administered intranasally. or in a suitable aerosol container for administration either orally or by inhalation. Inhalation Further examples of compositions for administration include aerosol containers with a capacity of 15 to 20 ml. 10 mg of monoclonal antibody generated against epitope 417-438 was added to ethanol (6-8m) and 0.1-0.2% lubricant, e.g. rubate 85 or oleic acid and such mixture is combined with a propellant, e.g. Freon, preferably (1,2 dichlorotetrafluoroethane) and difluoroc Dispersed in combination with lolomethane for either intranasal or oral inhalation administration. Fill a suitable aerosol container.

In humans in need of prophylactic or therapeutic treatment of R5V infection, this 417-438 epitope administered by inhalation to carry out treatments such as The preferred daily dosage to be adopted for the monoclonal antibodies produced is or about 0.1 mg/kg to about 10 mg/kg.

"Human" FIG.3 1 human-) Area specific to humans FIG. 4 1.4- □ 0.000+00 0.010000+, 000000+00.00000 0 antibody concentration (u9/m1) FIG. 6A Submission of translation of written amendment (Article 184-8 of the Patent Act) March 1993 zl fee

Claims (52)

[Claims] 1. in the light and/or heavy variable domain of the acceptor monoclonal antibody. At least some portions of the complementarity determining regions (CDRs) are linked to one or more donors. ・Replaced by a similar part of the CDR from the monoclonal antibody, To preserve the binding specificity of the acceptor monoclonal antibody, The light and/or heavy variable domain framework regions of a clonal antibody are minimally modified. Such donor antibodies, which may be modified or unmodified, are A modified antibody characterized by having specificity for an organism. 2. The antibody according to claim 1, wherein the microorganism is human respiratory syncytial virus (RSV). 3. 3. The donor antibody is directed against the fusion (F) protein of RSV. antibodies. 4. 3. The antibody according to claim 2, wherein the donor antibody is directed against epitope 417-438. body. 5. A claim having the following N-terminal variable domain amino acid sequences in the heavy and light chains: Antibody according to 2. Heavy chain: [There is an array] Light chain: [Sequence available] 6. 3. The antibody of claim 2, wherein the donor monoclonal antibody is RSV19. 7. 3. The antibody of claim 2, wherein the donor monoclonal antibody is RSV20. 8. The antibody according to claim 6, which is HuRSV19VH/VK. 9. The antibody according to claim 6, which is HuRSV19VHFNS/HuRSV19VK. . 10. Claim 1, which is a Fab fragment or (Fab')2 fragment. Antibodies listed above. 11. comprising the modified antibody according to claim 1 and a pharmaceutically acceptable carrier or diluent. Pharmaceutical composition. 12. 12. The composition according to claim 11, wherein the microorganism is human RSV. 13. Claim 11 wherein the donor antibody is directed against the fusion (F) protein of RSV. Compositions as described. 14. 12. The donor antibody is directed against epitope 417-438. Composition of. 15. The engineered antibody contains the following N-terminal variable domain amino acids in its heavy and light chains: 12. The composition according to claim 11, having the sequence. Heavy chain: [There is an array] Light chain: [Sequence available] 16. 12. The composition of claim 11, wherein the donor monoclonal antibody is RSV19. . 17. The composition according to claim 11, wherein the donor monoclonal antibody is RSV20. . 18. 17. The composition according to claim 16, wherein the modified antibody is HuRSV19VH/VK. 19. Claim that the modified antibody is HuRSV19VHFNS/HuRSV19VK 16. The composition according to 16. 20. The modified antibody is a Fab fragment or (Fab')2 fragment The composition according to claim 10. 21. Claims 4, 5, 6, 7, 8 and 9 of effective human RSV infection inhibiting doses. administering any of the modified antibodies to a human in need of prevention of human RSV infection; The preventive nature of human RSV infection in such humans. 22. - a dose of about 1 mg/kg to about 20 mg/kg of the modified antibody parenterally, preferably 22. The method according to claim 21, wherein the method is administered intravenously or intramuscularly. 23. - Administer a dose of about 200 μg/kg to about 2 mg/kg of the engineered antibody intranasally. 22. The method according to claim 21. 24. Administration was started from the beginning of the RSV epidemic season (October-November), and 23. The method of claim 22, repeated every 6 weeks until the end of March-April. 25. Administration was started from the beginning of the RSV epidemic season (October-November), and 24. The method of claim 23, repeated every 6 weeks until the end of March-April. 26. At the beginning of the RSV epidemic, doses of engineered antibody from about 5 mg/kg to about 100 m g/kg intravenously or intramuscularly, or - a dose of about 0 ．． 22. The method of claim 21, wherein 5 mg/kg to about 10 mg/kg is administered intranasally. 27. Claim that the modified antibody is HuRSV19VHFNS/HuRSV19VK 21. The method described in 21. 28. 22. The method according to claim 21, wherein the modified antibody is HuRSV19VH/VK. 29. Claims 4, 5, 6, 7, 8 and 9 of an effective therapeutic dose for human RSV infection. Either engineered antibodies may be administered to humans in need of treating such infections therapeutically. A method for therapeutically treating human RSV infection in such a human comprising administering Law. 30. - a dose of about 2 mg/kg to about 20 g/kg of the modified antibody parenterally, preferably 30. The method according to claim 29, wherein the method is administered intravenously or intramuscularly. 31. A claim in which about 200 μg/kg to about 2 mg/kg of the modified antibody is administered intranasally. 29. The method described in 29. 32. Monoclonal antibody raised against epitope 417-438. 33. The monoclonal antibody of claim 32 and a pharmaceutically acceptable carrier or diluent. A pharmaceutical composition comprising: 34. The monoclonal antibody of claim 32 at an effective dose for inhibiting human RSV infection, comprising administering to a human being in need of preventing such infection; A method for preventing human RSV infection. 35. - a dose of monoclonal antibody from about 1 mg/kg to about 20 mg/kg parenterally 35. The method according to claim 34, wherein the method is administered intravenously, preferably intravenously or intramuscularly. 36. - a dose of about 200 μg/kg to about 2 mg/kg of monoclonal antibody intranasally 35. The method of claim 34, wherein the method is administered intravenously. 37. Administration was started from the beginning of the RSV epidemic season (October-November), and 36. The method of claim 35, repeated every 6 weeks until the end of March-April. 38. Administration was started from the beginning of the RSV epidemic season (October-November), and 37. The method of claim 36, repeated every 6 weeks until the end of March-April. 39. At the beginning of the RSV epidemic, -doses of monoclonal antibodies ~5 mg/kg ~ about 100 mg/kg intravenously or intramuscularly; 35. The antibody according to claim 34, wherein about 0.5 mg/kg to about 10 mg/kg of the antibody is administered intranasally. the method of. 40. an effective therapeutic dose of the monoclonal antibody of claim 32 for human RSV infection; comprising administering to a human being in need of treating such an infection; A method of therapeutically treating human RSV infection in humans. 41. - a dose of about 2 mg/kg to about 20 mg/kg of monoclonal antibody parenterally 41. The method according to claim 40, wherein the method is administered intravenously, preferably intravenously or intramuscularly. 42. Claim 40, wherein about 200 μg/kg to about 2 mg/kg of the antibody is administered intranasally. Method described. 43. 33. The monoclonal antibody according to claim 32, which is a Fab fragment. 44. 35. The method of claim 34, wherein the monoclonal antibody is administered by inhalation. 45. 41. The method of claim 40, wherein the monoclonal antibody is administered by inhalation. 46. 22. The method of claim 21, wherein the modified antibody is administered by inhalation. 47. 30. The method of claim 29, wherein the modified antibody is administered by inhalation. 48. Modified antibody variable regions required to produce modified antibodies with increased binding affinity A method of minimally modifying within the scope of the framework, comprising the following steps: (a) C Framework amino acids known to be important for interaction with DR Analyze the acids and find either a single framework amino acid or the corresponding amino acid from the same source as the CDR. (b) produced and tested modified antibodies that have been substituted with amino acids; (b) adjacent CDRs; The framework amino acids of the CDRs are analyzed and one or more amino acids within the four Engineered antibodies in which amino acids are replaced by corresponding amino acids from the same source in the CDRs (c) analyze the framework residues within the engineered antibody and amino acids have major differences in charge, size, or hydrophobicity from the same source of CDRs. Modified antibodies that have been substituted with the corresponding amino acids can be produced and tested. A minimal modification method consisting of: 49. A recombinant plasmid containing the coding sequence of the modified antibody of claim 1. 50. A mammalian cell transfected with the recombinant plasmid of claim 49. Cell strain. 51. A recombinant plus containing the coding sequence of the monoclonal antibody of claim 32. Mid. 52. A mammalian cell transfected with the recombinant plasmid of claim 51. Cell strain.