JPH06501840A - Multivalent anti-cytokine immunoglobulin - 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] Polyvalent anti-cytokinoimmunoglycerin σ purine field of invention The present invention provides multivalent anti-cytokine immunoglobulin and its production and treatment. Regarding use in medical treatment. In particular, polyvalent immunoglobulin is a Suitable for use in treatments where neutralization of the level of cytotoxicity is required.

Cytokines transmit signals from one cell to other cells of the same or different type Cytokines as used herein are a group of polypeptide mediators The term also includes lymphokines. For example, anti-cytokine immunoglobulin teeth.

Anti-tumor necrosis factor-α (TNF-α) or anti-tumor necrosis factor-β (TNF-β) It is an anti-inflammatory globulin and is used to reduce the level of TNF-α or TNF-β in the body. Can be used.

Background of the invention Antibodies, or immunoglobulins, are part of the 581 immunoglobulin class, immunoglobulins. phosphorus G (IgG), immunoglobulin M (IgM), immunoglobulin A (IgA) , classified into immunoglobulin D (IgD) and immunoglobulin E (IgE) . Each of these classes of immunoglobulins consists of two identical light polypeptide chains and two identical heavy polypeptide chains were linked to each other by disulfide bonds. Basic four-chain structure Consisting of This basic structure may hereinafter be referred to as an immunoglobulin molecule. Each immunity The globulin class is further divided into subclasses. For example, human IgG , consisting of subclasses IgG1, 1gG2, IgG3 and 1gG4. immunity A globulin's class and subclass are determined by its heavyweight type.

IgG is the main immunoglobulin in normal human serum and has the above-mentioned four-chain structure. It is a monomer. IgM is a smaller portion of the human immunoglobulin pool but.

Monomers with a four-chain structure account for a considerable proportion, and the disulfide bridges between the heavy chains the law of nature.

Also, the pentama connected by a polypeptide linker called a heavy chain = Mm has.

Among other immunoglobulins, IgA is the most common and can be found in monomeric or dimeric bases. It exists. IgD and IgE exist in extremely small amounts in humans, and eventually is always a monomer.

The amino-terminal domains of the basic heavy and light chains are characterized by sequence variability. They are called VH (variable heavy chain) domain and VL (variable light chain) domain. It will be done.

The vH and vL domains together form the antigen-binding domain of immunoglobulins. Ru.

That is, the basic four-chain immunoglobulin structure has 2@ antigen binding domains. death Therefore, IgM has a total of 10 antigen-binding molecules. After all, heaven Naturally occurring immunoglobulins react with IgG, IgD and IgD against the antigen they bind to. In the case of gE, 2. 2 or 4 for IgA. IgM has 10 and various valencies. will be shown.

Similarly, antibodies:! :! The antigen to be recognized may be one, one or many antigenic determinants or antigens. has an epitope. The antigenic determinants of a multideterminant antigen may be the same or different. In some cases For example, a small molecule or habten binds to the complementary antigen of one of the immunoglobulin molecules. It has a single antigenic determinant that is recognized by the binding region. In contrast, large proteins A molecule has a large number of determinants on its surface, for example as many as ten.

When an antigen with multiple epitopes exists, different determinants on the antigen can be recognized. The binding of two antigen molecules by the antibodies that recognize them is the sum of the bonds formed by each antibody. It is already well known that the bonus effect is always greater than By the way. For binding of antibodies to polymeric antigens with identical repeating determinants It is also known that the same thing can be said. In other words, the antibody-antigen interaction The apparent binding activity is 1 jI of the individual antigenic determinant-antibody combining site interaction! phase unit price Fa with 1 VH and 1 ■1 domain When changing from b-fragmentoto to divalent IgQ to pentamer IgM, multi-bond A significant increase in the strength of antigen-antibody complex formation occurs due to the bonus effect of titer (e.g. For example, -Essential 1mmunology-.

1, Roitt 6th edition, Blaekvell 5 scientific Pub 198g).

Various monoclonals against W tumor necrosis factor-α with the same vH tomeino compared the ability of anti-TNF antibodies (anti-TNF antibodies) to neutralize human recombinant TNF. In the course of testing, the present inventors discovered that the IgM antibody was observed to be significantly better. In addition, one monochrome against recombinant TNF Null IgG is stored for one year after production, aggregates into high molecular weight forms, and forms dimers and triglycerides. mer-containing antibodies exhibit higher neutralizing activity than newly produced monomeric antibodies. It was recognized that Also, dimer aggregates are slightly better than monomers The trimer gave even better neutralization. these observations From 1 the present inventors.

I tried crosslinking IgG that I had on hand. Cross-linking results in multivalent immunoglobulin, This resulted in a surprising increase in the neutralizing activity of the antibody.

Detailed description of the invention Therefore, the present invention is specific to the complementary site on the site force ino, A multivalent immunoglobulin comprising at least 3 gi of each antigen-binding domain/ Provide phosphorus.

Synthesis between antigen-binding tome and complementary cytokines/sites of multivalent immunoglobulins/ This interaction is a characteristic neutralizing effect. Cytokinesia measured in current It, in viLro or tn vivo tests Inhibit or reduce the biological activity of the drug! Taste.

Each of the at least three linked antigen-binding domains is linked to the same site. However, different antigen-binding domains in multivalent immunoglobulin Mains may be specific for different epitopes of the same cytokine , for example, each antigen-binding region has one neutralizing epitope of the cytokine it recognizes. however, it is compatible with the antigen-binding domain. As long as the overall interaction with the complementary site is neutralizing as described above, it is not necessary. It's not important.

At least 3 were connected. joins the same cytokino-specific antigen-binding domain. In addition, polyvalent immunoglobulins contain molecules that are specific for another cytokine or molecule. Alternatively, it may include two or more other antigen-binding domains.

The cytokine for which the polyvalent immunoglobulin of the present invention exhibits specificity is usually a soluble protein. It is a powerful input.

As an example of a cytokine for which the antibody-binding domain of multivalent immunoglobulin exhibits specificity tumor necrosis factor-α (DingNF-α), tumor necrosis factor-β (TNF-β or lymphokine), inotaleukin (IL-1, rL-2, rL-3, IL-4 , IL-5゜IL-6*), interferon (e.g. INF-α, INF -β, INF-γ, etc.) and colony stimulating factors (e.g. G-CSF, GM- CSpi) does.

Cytokines can be either monomers or antigen-binding domains of multivalent immunoglobulins. or has at least two epitope sites recognized by two or more Good too.

In addition, the antigen-binding domain of multivalent immunoglobulin is may be specific, i.e., the cytokine recognized is a polyvalent immunoglobulin. At least two recognized by one or more of the antigen-binding tomains of Robulin The cytokine, which may have an epitope site of gA, is For example, TNF-α may be the resultant multimer of any of the original It is known to exist in both gomer and monomer forms. That is, T.N. F-α molecules associate with each other to form trimers, thus forming multivalent immunoglobulins. can act like a multivalent binding partner to interferon -γ and TNF-β can also exist as oligomers.

The size of multivalent immunoglobulins is thought to be limited by solubility considerations. In other words, the number of linked antigen-binding domains is The complementing site should be carefully prepared to avoid forming an insoluble precipitate before contact with the ino. The upper limit of the number of linked antigen-binding domains is probably 40 However, the preferred number of linked antigen-binding domains/ For example, in the range of 4 to 20, preferably 4 to 10 antigen-binding tomains. It seems much smaller.

The antigen-binding tomeino of polymerized rabies grofurino is IgG, IgM, IgA, IgD or or derived from any class of IgH and any immunoglobulin subclass. Wear.

The immunoglobulin class of each antigen-binding tomain in a given polymerized immunoglobulin. or the subclasses may be the same or different; they are preferably all IgG It's a class.

Furthermore, all antigen-binding domains in multivalent immunoglobulins are derived from IgM. Temoyo L1. In that case, the polyvalent immunoglobulin may contain, for example, less than or equal to 10 or less. or in other respects as will become clear below. This is what natural IgM is. Similarly, all antigen-binding domains are IgA When derived from a polyvalent immunoglobulin, it is winged with the natural molecule. for example , the multivalent immunoglobulin contains 3 or at least 5 binding domains; or differ from natural IgA in other respects.

The antigen-binding domain of a multivalent immunoglobulin binds the whole immunoglobulin/molecule, e.g. 1g0 may exist as part of a molecule, and they are analogous to a complete molecule. even if it is a fragment of an immunoglobulin molecule produced by enzymatic digestion. good.

That is, the antibody binding domain is an IgG Fv, Fab, F@b' or F(ab)27 may be composed of lagumenoto, and furthermore, the binding site may be present on the corresponding 1 gM fragment.

Immunoglobulin molecules and fragments thereof are of animal origin, e.g. For example, it may be of mouse, rat, or human origin. They are, They may be polyclonal antibodies, but are preferably monoclonal antibodies. teeth.

It can be produced using well-known immunization techniques. For example, you can Tocaine is injected, the serum is collected, and purified or concentrated as appropriate, e.g., fixed. Affinity chromatography using conjugated cytokines as affinity medium The desired polyclonal antibody against the cytotoxic antibody is produced (by Alternatively, the spleen cells or lyphocytes can be extracted from the host injected with Cytocytokinesi. This is collected, for example, by the method of Galfre, G et al. (Nature, 266, 550.1977).

Once the resulting cells are isolated, monoclonal antibodies against the antigen are generated using conventional procedures. A monogenetic system for production is obtained. Antibody fragments can be prepared using conventional techniques. Ba.

For example, whole antibody Peven 7 (Parham: J, l@nuno1.+31 ．． 2895.1983) or Babai 7 (Lamoyi & N15onof f: J, 1mmuno1. Enzyme by Meth, 56.235.1983) can be produced by digestion Particularly useful immunoglobulins for use in the present invention include recombinant immunoglobulins and and its fragments are included. Especially useful for recombinant immunoglobulins.

(1) Antigen-binding domains, at least in part derived from different immunoglobulins Recombinant immunoglobulins that contain a protein, such as the hyperrecess of certain immunoglobulins or or complementarity determining regions (CDRs) of the second, winged immunoglobulin variable domain. ,centre.

−4ヮ−y+=g,7t recombinant immunoglobulin (European Patent Specification System 239 400).

(2) The nonvariable domain sequence is derived from another immunoglobulin. Recombinant immunoglobulin or fragment substituted by column (European patent specification) Showara 12 (1694, 125020, 171496, 173494 and 194 No. 276 (as described), and (3) substantially natural immunoglobulins. It has the structure of immunoglobulins, but the number of protein residues in the hinge region is similar to that of natural immunoglobulins. or the appearance of the recombinant immunoglobulin or fragment. One or more 7-stain residues in the face pocket are present in the native immunoglobulin. Recombinant immunoglobulin or fragment in place of other amino acid residues present (Respectively published International Patent Application No. WO39101974 and WO3 9101782).

is included.

More specifically, the 9 recombinant immunoglobulins are derived from 3 CDR grafts or 3 CDR grafts. It consists of immunoglobulins and their 7 fragments.

Recombinant immunoglobulins are whole immunoglobulin molecules or immunoglobulin molecule fragments. recombinant immunoglobulins and fragments as described above, and Single chain antibodies include single chain Fv.

The antigen-binding domains of multivalent immunoglobulins can also be linked by covalent cross-links. The bridge may directly link the antigen-binding domains (e.g., for multivalent Fv). (as described in PCT/GB 90100935), and also antigen binding. Large domain immunoglobulin fragments or whole immunoglobulin molecules If present as part of the crosslink, the bridge may be separate from the antigen binding domain.

Immunoglobulin molecules or fragments thereof containing antigen-binding tomains are Linked by disulfide bridges between in residues or via cross-licker molecules be done. Cross-linkers generally refer to the immunoglobulin molecules or molecules being linked. can be reactive with the side chains of amino acids in the segment. like this It is preferable that amino acids that are not directly involved in antigen binding are suitable for cross-linking. The appropriate amino acids include amino, sulfhydryl, and carboxylic acids that add cross-linkers. Boxil.

with side chains containing phenolic or aromatic or heteroaromatic functional groups It is an amino acid. Suitable amino acids include lysine, /stein, glutamic acid and or multitubular immunoglobulin oxidized carbohydrate residues, especially oxidized carbohydrate residues. suitable for cross-linking immunoglobulins and other proteins that can be linked via The child is well known to those skilled in the art. A list of suitable molecules can be found, for example: Pierce Cnemieal Company's 1989 Handboo k and (in the ieneral Catalog. Immunoglobulin molecules or One example of a method for crosslinking fragments or fragments is to link one of the components to a 2-imimide. one reacts with notiolano, the other reacts it with e.g. maleimide succinimide There is a method of activating with maleimide by reacting with tel.

As an alternative to covalent linkage, the antigen-binding domain can be linked e.g. are linked by interaction, and the linkage involves the immunoglobulin fragments or fragments to be crosslinked. are provided by antibody molecules that are specific to sites on the molecule. These sites are responsible for antigen binding. For example, when cross-linking whole immunoglobulin molecules (Ko(yo)

This allows for the use of anti-Fv antibodies attached to the constant region of the immunoglobulin to be linked. Therefore, it can be done.

An alternative method of achieving non-covalent cross-linking is to use biotin-derivatized immunoglobulins. A drug that causes molecules or fragments to aggregate through interaction with avinone. There is a method using othinoabinone crosslinking.

Regardless of the type of cross-linking used, antigen-binding tomains in multivalent immunoglobulins are Preferably in the correct orientation relative to the bond.

As previously mentioned, preferred polyvalent immunoglobulins of the invention include linked IgG moieties. children and/or IgG fragments. However, polyvalent immunity Loss/also. (can be based on 1 gM molecule or fragment) \　For example, polyvalent immunoglobulins can be obtained by e.g. partial enzymatic digestion. 7 fragments of natural IgM; alternatively, natural crosslinks of IgM may also be linked at other different positions and the whole immune 13M globulin molecule or Fragments (which can be cross-linked for two layers!) may be substituted to produce an artificial IgM, Furthermore, it is also possible to form decamers etc. by linking complete 1gM Beckmers. It is.

In a second aspect of the invention, a polymorphism of the first aspect of the invention for use in therapy is provided. Provide a titer of immunoglobulin Therapies for which the multivalent immunoglobulin molecules of the invention can be used include: It is desirable to reduce the level or inhibit the activity of cytokines present in V therapeutic potential. f Included Site Power Ino or Rinshi Kaino is 1g! There are people in circulation Even if it is

In addition, some substances may be localized at undesirably high levels in specific parts of the body. For example, elevated TNF-α levels are associated with immunomodulatory and inflammatory disorders and sepsis, Mapuko C! Associated with endotoxin//, and cardiovascular/thrombosis. T.N.F. -α (multivalent immunoglobulins with this specific antigen-binding domain may be septic) Ku: Yo Endo Toki 7//1 Tsuku, Adult Respiratory Distress Syndrome, AIDS, Allergies, Dry IU, T, B, inflammatory bone disorders, blood coagulation disorders, burns 11 organs or tissues Rejection phase after transplantation, as well as primary autoimmune diseases and organ-specific diseases such as thyroiditis. of conditions including chronic disease or non-specific organ diseases such as rheumatism and osteoarthritis. Can be used for treatment.

Furthermore, polyvalent immunoglobulin can alleviate the side effects associated with the development of TNF during the treatment of neoplasms. .

and treatment or prevention of graft rejection by the use of anti-lymphocyte antibodies/summer; It can also be used to eliminate or alleviate symptoms associated with depression.

Similarly, other cytokines such as interferon γ, IL-1, [L-2 , l The polyvalent immunoglobulin of the present invention having specificity for L-5 and IL-6 is 2 It can be used to treat conditions associated with elevated levels of problematic cytokines.

In one aspect of the present invention, one of the polyvalent immunoglobulins of the first nine aspects of the present invention.

Undesirable high levels of polyvalent immunoglobulin-specific cytotoxicity are involved Use in the manufacture of a medicament for the treatment of a condition is provided.

Preferably, the cytokine is interferonogamma (INF-7), 9-o IL-1 (IL-1), interleukin-2 (IT, -2), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and is tumor necrosis factor-β (TNF-β).

The medicament produced according to the third aspect of the invention may be in any suitable form in which it is used. The amount can be administered depending on the treatment being used. This medicine can be used for example to It is recommended that cytokines be used prophylactically in settings where elevated levels of cytokines are expected. After reaching a high level, or when the level is increasing, the site Used to lower levels of Cain.

According to yet another aspect of the present invention, the polyvalent immunoglobulins of the system of the present invention are .

Provides a pharmaceutical composition mixed with a pharmaceutically acceptable diluent, excipient or carrier do.

The composition may contain other active ingredients.The composition may include any suitable The dosage form may be in the form of an injection or infusion, e.g. A composition in a form suitable for parenteral administration, such as a single injection or continuous infusion, can be injected. For injection or injection, it is a polyvalent immunoglobulin in an oily or aqueous vehicle. in the form of a suspension, solution or emulsion, containing suspending agents, stabilizing agents and/or dispersing agents. Prescription agents such as agents can be included.

Alternatively, a composition may be in dry form for reconstitution with a suitable sterile liquid before use. Can also be done.

The dose when administering polyvalent immune globulin/globulins will depend on the nature of the condition being treated, the amount to be neutralized, etc. The extent to which the site power level has increased above the desired level or the extent to which it is expected to increase. Every time.

Whether polyvalent immunoglobulins are used prophylactically or to treat an existing condition The dosage may vary depending on the nature of the particular polyvalent immunoglobulin selected for treatment. Ta.

Selected depending on patient's age and condition.

Thus, for example, if a multivalent immunoglobulin has specificity for TNF-α, 1g0 molecule, 1g0 molecule is cross-linked with anti-Fv antibody, and multivalent immunoglobulin . Prevention or treatment of TNF-α related conditions such as inflammation or inflammation (If required, the appropriate dose is in the range of 0.001 to 10 g/kg/day.) It is surrounded. This dose provides relief from conditions associated with undesirable high levels of antigen. In still other embodiments of the invention, the invention may continue as long as necessary. treatment of human or animal subjects suffering from disorders associated with high levels of cytokines. provide therapeutic treatment methods; This method is.

An effective amount of the multivalent immunoglobulin according to the first aspect of the present invention is administered to a subject. It is.

In another aspect of the invention, one of the polyvalent immunoglobulins according to the first aspect of the invention is A manufacturing method is provided. This method uses immunoglobulin molecules or fragments thereof. For a suitable linker consisting of concatenating As mentioned above. That is, ligation connects immunoglobulin molecules or fragments. by reacting with covalent cross-linkers or by combining them with antibodies. It is understood that non-covalent cross-linking is effected by contacting the polymer with a flocculant.

In the case of one recombinant immunoglobulin, a method for producing multivalent immunoglobulin.

immunoglobulin molecules in suitable host cells transformed with suitable NA sequences. or expression of fragments.

Brief description of the drawing The invention will be explained below by way of example with reference to two drawings.

Figure 1 shows the residual activity of bioactive TNF-α after antibody treatment at +29/+6. I QI/4. Regarding the four emerging antibodies named CBS and 27/26, Figure 2 is a diagram plotted against the initial concentration of anti-TNF antibody. Figure 2 is a schematic showing how anti-TNF antibodies are linked by anti-Fc antibodies. (Although the array is illustrated as two-dimensional, it is actually three-dimensional.)

Figures 3-5 show the residual activity of bioactive TNF-α after antibody treatment with polyclonal anti- Antibodies - Three different antibodies cross-linked with Fe (101/4.HTNFI and CB S) plotted against the initial concentration of anti-TNF antibody.

Figure 6 shows the residual activity of TNF-α after antibody treatment with avidino/biotinylated Figure 7 is a diagram plotted against the initial concentration of biotinylated CBS antibody. Rifunatoki // Neutralization efficiency of antibody (hLT12Biotin).

The same antibody after avidino crosslinking (hL Tl2B 1olin A vidin ・Figure 8, which is a comparison diagram with 1), shows that the residual activity of TNF-α after antibody treatment is Figure 2 is plotted against initial concentration of bindingly cross-linked CBS antibody.

Figure 9 shows the plasmid diagram of plasmid pE20s6 containing the human Cμ coding sequence. Gram.

Figure 10 shows the residual activity of bioactive TNF-α after antibody treatment with the I of antibody 101/4. For gG and 1 gM virgin 1, the initial concentration of anti-TNF This is a diagram.

Numbers 2-5 indicate 4 separate samples of 101/4.

Antibodies 129/16 and +01/4 were raised against 9 recombinant human TNF-α. It is a mouse monoclonal antibody of immunoglobulin class IgG, onal In5 posture for Biological 5tanda Obtained from RDS and Controls (NTBSC). C BS and HTNF are also mouse monoclonal to human TNF-α. It is an antibody and was manufactured by Ceilteah. Antibody 27/26 to TNF-α It is a monoclonal IgM for Maxx and was obtained from NIBSC.

Comparisons were made using the L929 bioassay described. FJ In short, this is was carried out as follows.

L929 cells were produced using the following method.

2mM glutamine 10%FCS, and 100μg/ml gentamicin ( Adhesion in RP M I 1640 medium (Gibeo) containing RP 10) Supernatant medium was removed from L929 cells grown as a monolayer. Dulbecco's single layer Washed in A phosphate buffered saline solution CPBS (Gibeo). tripe Cells were removed from the plastic by adding SynEDTA (Gibco) for 10 minutes. cells were removed and the cell suspension was evaluated by Tripap Blue (Gibco) dye exclusion. A final density of 3×10 5 viable cells/ml was added to RP10 medium. this cell The 100 μm suspension was then transferred to a flat bottom 96-well microtiter tray (Falco n Microtest) and incubated overnight at 37°C. .

Dilutions of the antibodies to be tested are prepared in assay medium (Glutamino, Gettamy 7) and Prepared in RP MI 1640) containing 2% FCS and 1% actinomyne/D. Ta. A dilution of human recombinant TNF-α (NIBSC standard!) was also prepared in the assay medium. prepared and added to all dilutions. The L929 assay was then carried out as follows.

Remove RPMI medium by shaking the microtiter plate containing L929 cells. Place the 100 μm antibody and TNF# solution into a microtiter plate. Assay medium and the solution containing the highest antibody concentration were also added to the wells. The plates were then incubated overnight at 37°C.

- After incubating overnight, shake the L929 plate to remove the medium and inject once. Washed with PBS. Next, add methanol (AnalaR) to all wells. Add, leave it for 30 to 60 seconds, shake off the methanol, and remove the crystal violet. Add the solution (1% v/v in water) to the well and incubate for 5 minutes. Ta. This was then washed with running water, and the staining was carried out in a well with 100 μm of 30% water. Dissolved in acetic acid (AnalaR). Measures absorption at 570 μm and compensates for light scattering. 4100 absorptions were used as reference values for correction.

Surviving 929 cells take up crystal violet, but are killed by TNF. No uptake was observed in the treated cells, keeping the amount of TNF added to the well constant death.

In experiments in which antibody concentrations were varied, the absorbance of the extracted stain was associated with the killing of L929 cells. It can be used as the value of TNF in solutions used for.

Antibody 129/16.101/4. Results of experiments comparing CBS and 27/26 The results are shown in Figure 1. In this case, the TNF concentration in each well, 400 μg/1, is approximately It was sufficient to kill all cells. 6 The concentration of anti-TNF varied from well to well. From the results shown, significantly more antibodies than IgM antibodies were required to neutralize the same amount of TNF-α. It is clear that large amounts of IgG antibodies are required. For example, the starting concentration of TNF-α If the concentration is 400 μg/ml, the ID50 (antibody concentration that gives 50% inhibition) is For IgM antibody 27/26, it was 3.4 ng/ml, whereas for IgG anti- The 1Dso of the body is Sang/ml at +29/16 and 42℃g/ml at CB S. +l, +01/4 was llng/++l.

Therefore, IgM antibodies have significantly higher neutralizing activity than their IgG counterparts. It is clear that To determine whether the increased IgM neutralizing activity was a result of cross-linking of immunoglobulin units. In order to achieve this goal, various types of 1gGfc were cross-linked and the effects on neutralization activity were investigated.

Polyclonal anti-Fc antibody 0ackso specific for the constant region of the Mawas IgG molecule Selected IgGs were cross-linked using NS Labs). Anti-Fc antibody is antigen-binding IgG by its Fc region while retaining its binding tomeino which is utilized for It has a crosslinking effect. As shown in Figure 2, the binding mode is 1gG anti-TNF molecule. I by anti-Fc antibody molecules 7 within a sequence linked to the N-terminus of their constant region 5. The TNF molecule 9 placed is the C-terminus 6 of the anti-TNF molecule l! Antibody binding at +1 shown bound to the site. In this way, anti-TNF concentrations of 2 to about 2000 To perform cross-linking, where the children are cross-linked to each other, use the following operations: Each antibody to be tested was incubated with goat [F(ab')2] anti-mouse IgG-Fc. 9297 Nosei medium separately dissolved in 2 μg 7 ml antibody and 20°C. Up to 9 3-fold dilutions of the antibody-anti-Fc mixture prepared to a concentration of g/ml anti-Fc The dilutions were incubated at 37°C for 1 hour.

The neutralizing activity of cross-linked antibodies was determined using the L929 bioassay as described above. Tested. Add 100 μl of each antibody-anti-Fc mixture and TNF to a microtiter. Distributed into appropriate wells of a tartray. The concentration of TNF in each well is the same as before. It was set to 4009g/■1.

The effect of cross-linking is that the remaining TNF, which is used for cell killing, is transferred to the IgG antibody 101/4.　 Plotted against antibody dilution for each of HTNFl and CBS. This is clear from FIGS. 3 to 5. In each case, measurements were made using antibodies alone or anti-Fc. I added it.

Anti-Fe alone had no effect.

Figure 3 shows, for example, the effect of adding anti-Fe antibody to anti-TNF antibody +01/4. are doing.

This plot shows that at a given antibody dilution, the presence of cross-linking with anti-Fe clearly shows that more TNF is neutralized than in its absence. Ru.

Similar effects are shown in Figures 4 and 5 for antibody, HTNFI and CBS, respectively. is shown. The lower the neutralizing activity of the antibody in the absence of anti-Fc, the more It should be noted that the improvement in neutralizing activity brought about by the addition of Fc antibodies is significant. It is. Table 1 below lists antibodies +01/4. HTNFI and CBS respectively The effect of polyclonal anti-Fe on TNF neutralization by either In this case, neutralization was also performed to an efficiency close to the theoretical maximum, i.e. for antigen Points to the efficiency of anti-TNF antibodies in neutralizing TNF were improved up to a 1 molar excess of antibody. Summary of effects of reclonal anti-Fc Anti-TNF Anti-TNF improvement Single + anti-Fc double Excess molar amount of antibody required for 50% inhibition of TNF CB6 60 0.4 150 [al anti-TNF antibody CB6, e.g. logy, L, Hudson & F, VHay, 2nd 3rd edition, +9119. 　Blackwell　5cIent Mountain c　Listed in Publica+1ons biotinylation according to conventional methods.

The biotino-labeled antibody was mixed with Strebtoavino 7 (Jaekson Labs) and 3 Mixed in a molar ratio of 10:1, 11 and 10, then incubated at 37°C for 1 hour. A 3-fold dilution series of biotin-labeled antibodies and complexes that were incubated to form complexes. Columns into L929 assay medium. INL, perform L929 assay as described above. It was.

The results are shown in Figure 6. As is clear from this, biotinylated CBS and avidino were combined at 1 When complexed at a ratio of A complex formed by otinated antibody and avinone at a ratio of lO:1 or 1:10 Compared to.

Yes! It increased to

[b] The experiment in part [++] was crosslinked with avidino at a molar ratio of 1·l. The results were repeated using a biotinylated anti-rhinophokine antibody (hLT12). 7. As is clear from this, the neutralizing activity of the cross-linked antibody is due to the unconjugated biotinylated C3S, which was chemically cross-linked relative to the antibody, was prepared as follows.

165 g of C3S (5 mg/ml in PBS) was dissolved in 3.2 g of ethanol in 1 ml of ethanol. Incubate with iminothiolane B for 30 minutes at room temperature with gentle stirring. 4, 75 mg of C3S (5 mg/l in PBS) was added to 0.195 1 g of maleimidohexanoylsuccinoimide ester and stir gently. In both cases, remove excess reagent and incubate for 45 min at room temperature. CBS iminothiolane and male were removed by chromatography on an IO column. The imide derivatives were then incubated together in a ratio of 1 l for 2 hours at room temperature. Ethylmaleimide in DMSO was added to each Inno + Jube/1 solution reacted by ! ! The final JPO, 01mg of the reaction product was subjected to HPLC, and 9 fractions of 280++ The absorbance at ++ was used to determine the protein-containing fraction. and used it for L929A and Sei as mentioned above. The results of the crosslinking experiment are shown in Figure 8. Fraction: 6 corresponds to the monomer, and 9 fractions = 5 to 1. Contains an increased molecular weight of the antibody (the fraction Nia is smaller than the monomer In general, chemical cross-linking of antibody molecules occurs within the antibody. It is clear that this results in an increase in fG activity.

Practice Production of recombinant chimeric IM In yet another experiment, a recombinant chimeric IgM anti-TNF antibody was produced! 2 built, L929. The ability to neutralize TNF was tested in a q-ioassay.

Encodes the light and heavy variable domains of mouse IgG anti-TNF antibody 101.4. The human μ-heavy constant region (Cμ) was coded with the DNA of It was ligated with the DNA to be coded.

101, 4! ■Construction of M version of the chain Human Cμ DNA has the following coding sequence: Obtained as an Xbal Fragmenote placed as shown on the restriction map ( Ward, C, T et al., International lIImunology l, No.

3, 296-309.19119) Xba [fragment pEE? , HcMV (Stephens, P. & Cockett, M., 1. , Nucl, Ac1d, Re517, 7110.1 989) into the Xba site of plasmid pE205 as shown in Figure 9. 6 was manufactured. pE2056 is located at the beginning of the Cμ process. #Bsu It has only one restriction site for 361.

c==7: This TCA GGG, AGT, GCA, TCC i.e. pE20s 6 with HindIlltO and Bsu361? keratinized, large fragments, H ind [ll-B su consisting of the food sequence of 1014 heavy chain variable domains/ The following sequence ligates with 361 and crosses the V-Cμm junction of the new gene. was manufactured.

GGG, ACT, CTG, GTC, ACT, GTC, TCC, TCA, , GGG, AGT, GCA, TCC release, pE20sg~pE206 Samples 2 to 51i in Figure 10 each have 9 pairs in the plus midphase. Replacement chimera (g) The product is smaller than the mouse IKG product ,2 ■ ■ FIG.6 Avidin crosslinking of biotinated CB6 Residual TNF (I) g/m1) CB6 dilution (ng/m+) -C86 Zetin CB6-Bio:Avid lo:i-day-C60-Bio: Avid Ill-CBfyBio:Avid l:loFIG, 7 Bithione cross-linked oligomer hLT12 Residual lymphotoxin pg/ml [hLT12]ng/m+ FIG.9 Procedural amendment form) 1. Display of incidents Multivalent anti-cytokine immunoglobulin Full name (name Celtic Limited 4. Agent 6 - Number of claims increased by amendment 7- Subject of correction translation of the drawing Translation of drawings (page document (no change in content) international search report 1111,,,lli6MII Aetllell16# N6 PCT/Ta9 11012161++s, +1.1+s*s□,□101,611110PCT /Yari 91101216 International Investigation Report ;Work', 2:22;-2;"-゛.;1;77::koj,2ied',*　ζ; ;";::2 2° 2° 7; 6; 琵;; 7; 2F 77: 2G:゛""(°10 1"°1"Garbage 22 to pull T""River 1"°Pile 11ThellI#tIIn P#le+Ie mountain (轡1-＾Ti11MMaIll佽-@letllIMel1M lcuLjM+ThENselfn++w高吝Q1V啼*+61+hs*w-09mm Continuation of lfilsrm toge eR front page (51) Int, C1,5 identification symbol Internal office serial number // (C12P 211 08 C12R1:91) (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IT, LU, NL, SE), 0A (B F, BJ, CF, CG, CI, CSi, GA, GN, ~Dori, MR, SN, TD , TG), AT, AU, BB, BG, BR, CA, CH, C3, DE, DK.

ES, FI, GB, HU, JP, KP, KR, LK, LU, MC, MG, MN, MW, NL, NO, PL, R○, SD, SE, SO, US I

Claims (1)

[Claims] 1. Consisting of at least three linked antigen-binding domains, each domain 2. is a multivalent immunoglobulin that is specific for the complementary site of the cytokine. immunoglobe The binding interaction between the antigen-binding domain of phosphorus and the complementary cytokine site is a neutralizing interaction. 3. The polyvalent immunoglobulin according to claim 1, which is used for TNF-α, TNF-β, specific for interleukins, interferons, or colony stimulating factors Multivalent immunoglobulin according to "Claim 1 or 2" 4. Naturally as a multimer The method according to any one of claims 1 to 3, which has specificity for existing cytokines. ``Claim 1'' consisting of 5.4 to 20 antigen-binding domains of the polyvalent immunoglobulin The polyvalent immunoglobulin according to any one of 6. to 4. All antigen-binding domains are 7. The polyvalent immunoglobulin according to any one of claims 1 to 5, which is ras IgG. All antigen-binding domains are of class IgM and are distinct from natural IgM molecules. 8. The polyvalent immunoglobulin according to any one of claims 1 to 5. recombinant immunoglobulin The polyurethane according to any one of claims 1 to 7, consisting of brin or a fragment thereof. titer immunoglobulin9. Claim 1: The antigen-binding domains are linked by covalent crosslinks. 10. Antigen binding domain is non-covalent The multivalent immunoglobules according to any one of claims 1 to 8, which are linked by a binding interaction. Rin 11. Multivalent immunoglobulin 12 according to claim 1 for therapeutic use ．． Undesirable high levels of cytokines for which polyvalent immunoglobulins exhibit specificity The polyvalent immunization according to claim 1 in the manufacture of a medicament for the treatment of conditions involving Use of globulin 13. The polyvalent immunoglobulin according to "Claim 1" is pharmaceutically acceptable. Pharmaceutical compositions formulated with diluents, excipients or carriers 14. comprising administering an effective amount of the multivalent immunoglobulin according to "Claim 1" Humans or animals suffering from disorders involving undesirably high levels of cytokines Method of therapeutic treatment of the subject 15. Immunoglobulin molecules or fragments thereof as appropriate The production of multivalent immunoglopurins according to claim 1, which are linked together with a linker. Method