KR20000075545A - Gamma-1 and gamma-3 anti-human cd23 monoclonal antibodies and use thereof as therapeutics - Google Patents

Abstract

The present invention relates to monoclonal antibodies which specifically bind to low affinity receptors for human CD23, IgE (FceRII / CD23) and contain human gamma-1 or human gamma-3 constant domains. Such antibodies are useful for regulating or inhibiting induced IgE expression. Thus, such antibodies have practical use in the treatment or prevention of diseases in which inhibition of induced IgE production is therapeutically desirable, including allergic diseases, autoimmune diseases and inflammatory diseases.

Description

Gamma-1 and gamma-3 anti-human CD23 monoclonal antibodies and methods of using them as therapeutic agents {GAMMA-1 AND GAMMA-3 ANTI-HUMAN CD23 MONOCLONAL ANTIBODIES AND USE THEREOF AS THERAPEUTICS}

IgE is a member of the family of immunoglobulins that are widely applied throughout the general population by mediating allergic reactions such as asthma, food allergies, type 1 hypersensitivity and well-known sinus inflammatory allergic rhinitis and conjunctivitis. IgE is secreted by B-cells and expressed on the surface of these cells. IgE synthesized by B-cells can be immobilized on the B-cell membrane by short transmembrane domains bound to mature IgE sequences. Membrane and secreted variants of IgE are formed in small cells by differential splicing of IgE RNA copies.

IgE also binds to B-cells (and T cells, leukocytes, Langerhans cells, dendritic cells of vesicles, natural killer cells, eosinophils and platelets) via the Fc region, thereby lowering the affinity IgE receptor (FceRII, hereinafter “FCEL”). ) And bind to mast cells and basophils through the Fc region to become high affinity IgE receptors (FceRI, hereinafter “FCEH”) Low affinity IgE receptors are generally referred to in the literature as CD23.

When mammals are exposed to allergens, antigen presenting cells process antigens for presentation to helper T cells. These helper T cells secrete cytokines, such as IL-4, which help B-cells to amplify clonal and secrete more allergen specific IgE. This newly synthesized IgE is then released into the circulation, which binds to mast cells and eosinophils via high affinity receptors on the cell surface. These mast cells and eosinophils are thereby sensitive to specific allergens. Exposure to the same allergen then binds to specific IgE and eosinophils on the surface of the mast cells to crosslink FceRI on these cells to activate the release of histamine and other factors that cause clinical hypersensitivity and anaphylaxis.

Antibodies that can bind to IgE that can bind to FCEL (CD23) but not to FCEF have been reported in the literature (see, eg, WO 89/00138 and U.S.). Patent 4,940,782. These antibodies are described as clinically advantageous because they bind to IgE that binds to a low affinity receptor (FCEL) or to cyclic IgE but not to IgE that binds to a high affinity receptor (FCEH). Thus, these antibodies will not activate mast cells or eosinophils.

Moreover, anti-CD23 antibodies have been reported to serve as therapeutics for, for example, allergic diseases, inflammatory diseases and autoimmune diseases. For example, WO 9612741, Bonnefoy et al, reports that ligands that bind CD23, such as monoclonal antibodies, are useful for the treatment or prevention of inflammatory diseases, autoimmune diseases and allergic diseases.

The use of monoclonal antibodies against CD23 as both IgE agonists and antagonists has been reported. IgE antagonists have been reported to have potential use in the treatment of diseases or diseases for which IgE inhibition is therapeutically desirable, such as allergic rhinitis and conjunctivitis, atopic dermatitis and asthma. For example, WO 8707302 (1987) to Bonnefoy et al., Which is clearly useful for assaying the presence of IgE receptors on cell types, monoclonal antibodies against CD23 in humans as therapeutics in diseases where regulation of IgE is therapeutically desirable. Is reported.

In part, because of their potential as therapeutic or diagnostic agents, several groups have reported the development of monoclonal antibodies against CD23 (see, eg, Rector et al., Immunol., 55: 481-488 (1985); Suemura et al., J. Immunol., 137: 1214-1220 (1986); Noro et al., J. Immunol., 137: 1258-1263 (1986); Bonnefoy et al., J. Immunol., 138: 2170-2178 (1987); Flores-Romo et al., Science, 261: 1038-1046 (1993); Sherr et al., J. Immunol., 142: 481-489 (1989); and Pene et al., Proc. Natl. Acad. Sci., USA, 85: 6880-6884 (1988). Moreover, as described above, the use of such antibodies in the specific inhibition of IgE production in systems in which cytokine (IL-4) is induced due to IgE synthesis has been reported [Flores-Romo et al ( Id.); Sherr et al. (Id.); Bonnefoy et al. (WO 8707302); Bonnefoy et al. (WO 8707302); Bonnefoy et al. (WO 9612741); Bonnefoy et al., Eur. J. Immunol 20: 139-144 (1990); Sarfati et al., J. Immunol 141: 2195-2199 (1988) and Wakai et al., Hybridoma 12: 25-43 (1993). In addition, Flores-Romo et al. (Same author) describe that Fabs prepared from anti-CD23 antibodies inhibit antigen specific induced IgE responses in rats in vivo. However, although reported, the mechanism by which anti-CD23 antibodies modulate IgE expression and in particular how they block IL-4 induced IgE production is still unclear.

Anti-CD23 antibodies have been shown to inhibit IgE production by signaling through CD23 present on the surface of IgE-secreting B cells. The action of unregulated CD23 on IgE-secreting B cells has been suggested to be a feedback suppression of IgE production. Nature 369, 753-756 (1994). This suggestion was theorized because mice in which the CD23 gene is removed are increased compared to the control and the IgE is continuously produced [Yu, et al.]. In addition, binding to CD23 by the IgE complex or by monoclonal antibodies to anti-CD23 has been reported to inhibit ongoing IgE synthesis by lymphoblastic cell lines that constitutively secrete IgE [Sherr et al., (Id.)]. This appears to be due to the downregulation of messenger RNA against these intracellular secreted IgE heavy chains (Saxon et al., J. Immunol., 147: 4000-4006 (1991)). However, the exact mechanism by which IgE expression is inhibited has not yet been accurately described in systems where IgE secretion is IL-4 induced.

Cross-linking of surface Ig and Fc gamma RII on B cells (B cell receptor) has also been reported to induce down regulation of Ig expression [D'Ambrosia et al., Science, 268: 293-297 (1995). )]. Similar mechanisms can be proposed for B cells that secrete IgE that also have cell surface CD 23 and Fc gamma RII. Anti-human CD23 antibodies that are bound to cells by antigen and also to Fc gamma RII via Fc interaction can send signals that inhibit IgE secretion via Fc gamma RII.

Mechanisms associated with IgE inhibition by anti-CD23 antibodies have been proposed to include blocking interactions other than the interaction between membrane CD23 and IgE. In this regard, CD23, a member of the C-type lectin family, has been shown to interact with several other ligands, such as CD21, CD11b and CD11c, present in various cell types, including T cells and leukocytes. In this regard, CD23 can be thought of as a cell adhesion molecule.

Thus, it has been suggested that the interaction of CD21 with CD23 may be involved in antigen presentation and subsequent IgE production. The model suggests CD23 B cells that bind to CD23 on activated T cells, predominantly present in atopic individuals, and then send an activation signal for IgE production [Reconant et al., Immunol., 88: 35-39. (1996); and Bonnefoy et al, Int. Amer. Allergy Immunol., 107: 40-42 (1995). Blocking this interaction with anti-CD23 could block induced IgE production [Aubry et al., Nature, 358: 505-507 (1992) and Immunol., 5: 944-949 ( 1993); Grosjean et al. (1992); Bonnefoy et al., Curr. Opin. Eur. J. Immunol., 24: 2982-2988 (1994); Henchoz-Lecoanet et al., Immunol., 88: 35-39 (1996) Nambu et al., Immunol. Lett., 44: 163-167 (1995); Bonnefoy et al., Int. Amer. Allergy Immunol., 107: 40-42 (1995). It is also possible that antigen presentation is upregulated by CD23 on antigen presenting B cells that bind to CD21 on T cells.

Another mechanism that will likely explain the effect of CD23 on IgE production is related to the soluble form of CD23. CD23 has been reported to divide from the cell surface releasing several different forms of soluble CD23 or IgE binding factors (Sarfati et al., Immunol., 53: 197-205 (1984)). Soluble CD23 is a cytokine, one of its reported activities is an increase in IL-4 induced IgE production from B cells [Ref. Pene et al., J. Cell Biochem., 39: 253-269 (1989) ); Pene et al., Eur. J. Immunol., 18: 929-935 (1988); Sarfati et al., J. Immunol., 141: 2195-2197 (1988); Sarfati et al. (1984) (Id.); Saxon et al., J. Clin. Immunol. Allergy, 86 (3 pt 1) 333-344 (1990)]. In addition, certain forms of soluble CD23 have been reported to inhibit IgE production (Sarfati et al., Immunol., 76: 662-667 (1992)). Thus, anti-CD23 antibodies can block IgE production by 1) inhibiting the IgE increasing effect of soluble CD23 and / or 2) blocking the proteolytic release of soluble CD23 from the cell surface.

As such, based on the foregoing, the prior art is quite complex and unclear in terms of the effects of CD23 on the action and IgE production more specifically, and the means by which specific ligands affect IgE production. Do.

The present invention relates to monoclonal antibodies that specifically bind to low affinity receptors for human CD23, IgE (FceRII / CD23) and contain human gamma-1 or human gamma-3 constant domains and methods of using them as therapeutic agents. It is about.

1 is a graph comparing the in vivo IgE inhibitory activity of murine anti-human CD23 monoclonal antibody (MHM6) with anti-human CD23 monoclonal antibodies (5E8, 6G5, 2C8, B3B11 and 3G12) of five primates.

FIG. 2 shows the primate monoclonal antibodies 5E8 and 6G5 bound to epitopes on human CD23 distinguished from the commercial murine antihuman CD23 monoclonal antibody MHM6 (middle panel of FIG. 2), competing with each other (in FIG. 2). Bottom panel). Primates anti-human CD23 monoclonal antibodies 2C8 and B3B11 compete with MHM6 (top panel in FIG. 2).

FIG. 3 is a graph comparing the in vitro IgE inhibitory activity of a particular primate's antihuman CD23 monoclonal antibody 5E8 with four different primateized versions of said primate monoclonal antibody. The sequence is as follows:

p5E8G4P—This primatized antibody contains a human kappa light chain constant region, and a human gamma 4 constant region containing P variants. Angal et al., Mol. Immunol., 30: 105-108 (1993);

p5E8G4PN—This primatized antibody contains a human kappa light chain constant region, and a human gamma 4 constant region with P variants. Angal et al., Mol. Immunol., 30: 105-108 (1993). Such antibodies also contain variants in the heavy chain variable region that change asparagine residues (potential carbohydrate attachment sites) to lysine;

P5E8G1—This primatized antibody contains human kappa light chain constant region and human gamma 1 constant region;

p5E8G1N-These primatized antibodies contain human kappa light chain constant regions and human gamma 1 constant regions. Such antibodies also contain variants in the heavy chain variable region that change asparagine residues (potential carbohydrate attachment sites) to lysine.

4 is a table comparing the apparent Kd (nm) of the antibodies identified in FIG. 3 and summarizing their IgE inhibitory activity.

FIG. 5 is a graph comparing the in vitro IgE inhibitory activity of specific primates of antihuman CD23 monoclonal antibody, 6G5 and two different primateized versions of 6G5, with the sequence as follows:

p6G5G1-such primatized antibodies contain human lambda light chain constant regions and human gamma 1 constant regions;

p6G5G4P—This primatized antibody contains a human lambda light chain constant region and a human gamma 4 constant region with P variants. Angal et al., Mol. Immunol., 30: 105-108 (1993).

FIG. 6 is a graph comparing in vitro IgE inhibitory activity of F (ab ') ₂ derived from primate anti-human CD23 monoclonal antibody 2C8 and 2C8.

FIG. 7 is a graph showing against inhibition of in vitro IgE activity of F (ab ′) _divalent primate antihuman CD23 monoclonal antibody 2C8 derived from 2C8. FIG.

FIG. 8 is a graph showing in vivo IgE inhibitory activity of specific human primate anti-CD23 monoclonal antibody, 5E8, in an SCID animal model.

FIG. 9 is a graph comparing the in vivo inhibitory activity of p6GSG4P of antihuman 6G5 and their primateized versions of primates.

10 is a graph showing the in vivo IgE inhibitory activity of p6G5G1 of primate antihuman CD23 monoclonal antibody 6G5 and their primateized versions.

Accordingly, it is an object of the present invention to prepare novel ligands (antibodies) specific for CD23, and to describe the mechanism by which anti-CD23 antibodies modulate IgE expression using such antibodies.

Another object of the present invention is to prepare novel ligands (antibodies) that bind to CD23, in particular human CD23, which have an improved ability to inhibit induced IgE expression.

Still another object of the present invention is to prepare an anti-human CD23 antibody containing human gamma-1 or human gamma-3 constant domains.

Another object of the present invention is to prepare multivalent antihuman CD23 antibodies which may be more effective by increased potency against crosslinked CD23 and Fc receptors.

Another object of the present invention is to provide a pharmaceutical composition containing an anti-human CD23 monoclonal antibody comprising a human gamma-1 or gamma-1 constant domain capable of inhibiting induced IgE production.

Another object of the present invention is to use an anti-human CD23 monoclonal antibody comprising a human gamma-1 or human gamma-3 constant domain for the treatment or prevention of a disease in which inhibition of induced IgE production is therapeutically desirable. have.

More particularly, it is an object of the present invention to treat or prevent allergic diseases, autoimmune diseases and inflammatory diseases using antihuman CD23 monoclonal antibodies comprising human gamma-1 or human gamma-3 constant domains.

Definition of terms used herein

Chimeric Antibodies:

Two different antibodies, usually antibodies of different species, most typically a recombinant antibody containing regions from rodent variable sequences and human constant domain sequences.

Antihuman CD23 Gamma 1 Antibody

An antibody that specifically binds to human CD23 containing human gamma 1 constant regions or fragments, or variants thereof that inhibit induced IgE production. Such antibodies are particularly rodent or primate variable domains or antigen binding moieties comprising human gamma 1 constant domains, fragments, or variants thereof that inhibit in vitro-induced IgE production, humanized primateization and human antihuman Antibodies containing CD23 monoclonal antibodies.

Antihuman CD23 Gamma 3 Antibody

An antibody that binds to human CD23 containing human gamma 3 constant regions, fragments, or variants thereof that inhibit induced IgE production. Such antibodies are, in particular, rodent or primate variable domains or antigen binding moieties comprising human gamma 3 constant domains, fragments, or variants thereof that inhibit in vitro-induced IgE production, humanized primateization and human antihuman Antibodies containing CD23 monoclonal antibodies.

Variants of Antibody Constant Domains

An antibody according to the invention which contains variants, substituents or deletions of the constant region and which can preferably change the level of efficacy, ie FcR binding, without altering the basic effector action mediated by the constant region.

Primateized Antibodies

A recombinant antibody containing a primate variable sequence or antigen binding portion, and a human constant domain sequence.

Print antibody

By modifying the antibody variable region of a human to approximately mimic a variable region or antigen binding portion other than human that eliminates or reduces potential immunogenicity when administered to a human without abolishing the specificity or affinity of the immunoglobulin. Containing a recombinant antibody. There are several known printing methods, including “veneering” which involves selecting modifications of surface residues, framework replacements, (CDR implantation), and molecular modeling.

Gamma 1 constant domain:

Certain forms of constant domain sequences that confer activity on antibody specific effectors. In the present invention, a gamma 1 constant domain refers to a human gamma 1 constant domain, fragment thereof, which retains gamma 1 effector function in the form of an anti-CD23 variable domain sequence or antigen binding moiety. Variants include human gamma-1 constant domains comprising deletions, replacements, or additions of one or more amino acid residues. This effector function is manifested by the ability of antibodies containing these constant domains to inhibit induced IgE production.

Gamma 3 constant domains:

Certain forms of constant domain sequences that confer activity on antibody specific effectors. In the present invention, a gamma 3 constant domain refers to a human gamma 3 constant domain, fragment or variant thereof that retains gamma 3 effector function in combination with an anti-CD23 variable domain sequence or antigen binding moiety. Variants include human gamma-3 constant domains comprising deletions, substitutions or additions of one or more amino acid residues. This effector function is manifested by the ability of antibodies containing these constant domains to inhibit induced IgE production.

CD23:

This is referred to as the low affinity receptor for IgE, FceRII / CD23.

Anti-CD23 Antibodies:

An antibody that specifically binds to CD23, preferably human CD23.

Detailed description of the invention

As mentioned above, although a number of groups have reported the production of anti-CD23 antibodies and their use as antagonists and modulators for regulating IgE production, such antibodies in the system in which IL-4 modulates IgE production are called IgE. The mechanisms regulating expression are still unclear. As such, it would be advantageous if such an antibody means that the means for regulating IgE expression can be clearly explained, or at least more specifically, because such information may be useful for the treatment of a disease when modulation of IgE production is therapeutically desired. It will be potentially useful for designing therapeutics. In particular, it would be advantageous if improved antibodies were obtained that were specific for CD23 with improved ability to inhibit induced IgE production, since increased IgE levels were associated with many disease processes, such as allergic diseases, inflammatory diseases and It is believed to be associated with autoimmune diseases. Examples of such diseases are atopic dermatitis, eczema, allergic rhinitis and conjunctivitis, Job's syndrome, and asthma.

In this regard, the inventors have surprisingly found that anti-human CD23 monoclonal antibodies containing human gamma-1 constant domains are free of effector types that differ in IgE production, such as human gamma-4 constant domains or CD23 monoclonal antibodies or effector functions. It has been found that it inhibits IgE production in systems induced by IL-4 which are significantly better than CD23 monoclonal antibodies of the type comprising antibody fragments. Antihuman CD23 monoclonal antibodies containing human gamma-3 constant domains are also included, since human gamma-3 constant domains have been shown to mediate effector functions such as human gamma-1.

There are currently five defined effector functions for the IgG (gamma) class of antibodies. Two of these actions, complement activation and FcγRN interactions were not found in the in vitro assays described herein, and therefore seem to be unrelated to the molecular mechanism. The remaining three FcγR receptors have been shown to interact with IgG class antibodies: FcγRI, FcγRII (containing at least 6 different proteins) and FcγRIII (containing at least two different proteins). All three receptors interact with both IgG1 and IgG3.

FcγRI is the only of the three classes of receptors with acceptable affinity for IgG. The antibody binds to both 5-monomer gamma-1 and gamma-3 with K _a of about 5 × 10 ⁸ M ⁻¹ . However, this affinity for human gamma-4 is about 10 times less and does not bind to human gamma-2 at all. Fries et al., 1982, J. Immunol. 129: 1041-1049; Kurlander and Batker, 1982, J. Clin. Invest. 69: 1-8; Woof, 1986, G. Mol. Immunol. 21: 523-527; Burton and Woof, 1992, Human Antibody Effector Function, Adv. Immunol. 51: 1-84.

Although the affinity of human FcγRII and FcγRIII to human IgG is generally very small (K _a <10 ⁷ M ⁻¹ ), the affinity for human IgG1 and human IgG3 increases significantly when they bind to the antigen. (K _a ?? 2 to 5 × 10 ⁷ M ⁻¹ ) (Karas et al., 1982, Blood 60: 1277-1282). The affinity of human FcγRII for human IgG2 bound to the antigen has a given conflicting result. Human FcγRIII does not bind to human IgG2. Human FcγRII and human FcγRIII do not bind to human IgG4 [Ref. Van de Winkel and Anderson, 1991, J. Leuk. Biol. 49: 511-524; Huizinga et al., 1989, J. Immunol. 142: 2359-2364.

Although Fc mediated effector function is sometimes important for the therapeutic activity of antibodies, this finding is surprising for anti-CD23 antibodies because the role of effector functions in the IgE inhibitory activity of anti-CD23 antibodies has previously been reported. Because nothing has happened. Indeed, previous evidence suggested that antibody effector function was not critical to the ability of anti-CD23 antibodies to inhibit IgE production. For example, Flores-Romo et al., Science, 261: 1038-1041 (1993) indicate that Fabs prepared from polyclonal anti-CD23 antibodies inhibit IgE antigen specific responses induced in vivo. Reported.

The fact that the effector function mediated through the constant region of the anti-CD23 antibody is clearly related is that we have isolated various primate antibodies specific for CD23 with anti-IgE inhibitory activity and have IL-4 in vitro and in vivo. Regarding their ability to inhibit induced IgE production, it was found after comparing these antibodies with primate versions. Although antibodies structured with human gamma-4 constant regions failed to inhibit IgE antigen specific responses in vitro, antibodies containing human gamma-1 constant regions were successful.

Since one (or more) of the three classes of FcγR receptors, FcγRI, FcγRII, or FcγRIII seems to be related to specific effector function mediated through the gamma-1 domain, and these classes of receptors are responsible for the gamma-3 domain It is logical that anti-CD23 antibodies containing gamma-3 domains will also be successful in inhibiting IgE antigen specific responses in vitro because they also bind to the containing antibody.

More specifically, and as described in more detail below, five primate monoclonal antibodies that specifically bind to both cell and soluble CD23 are commonly assigned Application No. 08 / 379,072, incorporated herein by reference. Isolated from Old World monkeys (short-tailed monkeys) according to the method described in (currently granted). The application discloses the desired antigens in Old World monkeys, means for generating monoclonal antibodies against the desired human antigens, and their advantages associated with antibodies of other species as therapeutics, eg, humans and Old World monkeys. It describes in detail the benefits of reducing or potentially eliminating immunogens in humans because of their phylogenetic proximity. Indeed, due to the phylogenetic proximity of these species, it is difficult to distinguish Old World immunoglobulins from human immunoglobulins by sequence comparison.

Four of these five primate monoclonal antihuman CD23 antibodies have been shown to be able to inhibit IL-4 induced IgE production in the in vitro B cell assay described in detail below, the most potent antibody being SCID It has also been found to inhibit IL-4 induced IgE in mouse animal models (also described below). Based on this IgE inhibitory activity, and the anticipated low immunogenicity of humans, such antibodies are potentially suitable as therapeutic agents for treating diseases in which inhibition of IgE production is therapeutically desired.

However, to further reduce immunogenicity, two primate monoclonal antibodies (chimerized) according to the method also described in US patent application Ser. No. 08 / 379,072 (currently issued), incorporated herein by reference One type of antibody) was selected to be primateized (registered). Primateization (registration) refers to the generation of recombinant antibodies developed by IDEC Pharmaceuticals Corporation, which essentially comprises primate variable regions and human constant regions. Primateization of two primate antihuman CD23 monoclonal (5E8 and 6G5) antibodies with potent IgE inhibitory activity was performed to eliminate any latent immunogenicity attributed to primate constant domains in humans.

In addition, human gamma 4 variants of these specific antibodies were initially generated because of the inventors' first predictions from the literature that Fc effector function was not required for IgE inhibition. However, very surprisingly, the production of both gamma-4 variants from these primate monoclonal antibodies is ineffective, i.e. they are more primatized than primate antibodies that inhibit IL-4 induced IgE production in an in vitro assay. It was found that it requires a fairly high concentration of gamma 4 antibody.

Moreover, when the same two primate antibodies are converted to human gamma-1 variants (by replacement of primate constant domains by human gamma-1 constant domains), these gamma-1 antibodies are highly resistant to in vitro induced IgE production. The discovery of effective suppression is even more surprising. As such, our results suggest that it is clearly important for the ability of anti-human CD23 antibodies to inhibit induced IgE production. This assumption is based on the fact that the third anti-human CD23 monoclonal, i.e., 2C8 antibody, found by the inventors to inhibit IgE production in vitro was found to be able to substantially inhibit in vitro-induced IgE production. ab ') was identified when converted to ₂ . Indeed, such F (ab ') ₂ has been found to counteract the inhibitory effect on induced IgE blocking the activity of primate anti-human CD23 monoclonal antibody 2C8.

Furthermore, removing the glycosylation site in the heavy chain variable region of one of the antibodies (5E8) does not have any effect on binding of the antibody to CD23 (as evidenced by the obtained Kd value), or induced IgE inhibition. Turned out. As such, it was found that the difference in IgE inhibition was obviously independent of the glycosylation difference.

While primatized gamma 1 variants of primate 6G5 have been shown to inhibit IgE expression induced in SCID mice, primate 6G5 or primateized p6G5F4p at the same concentration did not inhibit induced IgE expression. Thus, antibodies containing human gamma-1 constant domains have been found to be much more effective in in vivo animal models than primate monoclonal antibodies. Moreover, the inventors have found that since the gamma-1 and gamma-3 domains have affinity for the same class of Fc receptors, anti-CD23 antibodies containing human gamma-3 constant domains can be combined with antibodies having gamma-1 constant domains. It is expected to be effective together.

Thus, based on these results, it was surprisingly found that the active Fc region, in particular the human gamma-1 or human gamma-3 region, is significantly associated with the IL-4 induced IgE inhibition mechanism by antihuman CD23 monoclonal antibodies. These findings are based in particular on early reports that Fabs derived from polyclonal anti-CD23 antibodies can inhibit induced IgE production, and various theories about how CD23 affects induced IgE expression. It is a very unexpected find based on.

Accordingly, the present invention relates to anti-human CD23 antibodies containing human gamma-1 or gamma-3 constant domains and methods of using them as therapeutic agents based on their ability to effectively inhibit IgE expression.

One skilled in the art can prepare antihuman CD23 antibodies containing human gamma-1 or gamma-3 constant domains by methods well known in the art for preparing chimeric antibodies. In essence, this method clones a hybridoma or cell line that produces an antihuman CD23 antibody in a desired host or in vitro, and produces an antihuman CD23 monoclonal antibody that exhibits the desired properties, eg, a suitable CD23 binding affinity. Cloning a nucleic acid sequence encoding such an antibody from the hybridoma or cell line by, for example, a polymerase chain reaction using a suitable primer, isolating the variable domain contained therein, and converting this variable domain into a human gamma Recombination into a −1 or gamma-3 constant domain and a suitable human light chain constant domain and cloning the chimeric antihuman CD23 gamma-1 or gamma-3 immunoglobulin in a suitable expression system to express the resulting nucleic acid sequence. Preferably, the anti-human CD23 antibody of the invention will have an apparent CD23 binding affinity of 0.1 nM to 1000 nM, more preferably at least 50 nM, most preferably at least 5 nM.

Suitable host cells for the expression of recombinant immunoglobulins are well known in the art. For example, recombinant antibodies include Chinese hamster ovary (CHO) cells, DG44 or DUXB11; Or CHO cell CHO K-1; Mouse myeloma cells SP2 / 0 or X63-Ag8.653 or NSO; Rat myeloma cells YB2 / 0; Hamster cub kidney cells, BHK; Human embryonic kidney cell line, 293; Monkey kidney cells, CV1; Human lung fibroblasts, WI38; Human cervical carcinoma cells, HELA; It can be expressed in insect cells, plant cells, yeasts or bacteria. In addition, vectors suitable for the expression of immunoglobulins are also well known in the art and are currently commercially available.

Particularly preferred vector systems include translations described in US patent application Ser. No. 08 / 147,696, now granted, which includes a translatingly damaged domain selectable marker containing an intron into which the desired heterologous DNA is inserted. It is a damaged vector system. Such vector systems have been found to provide high yields of recombinant proteins such as immunoglobulins. However, the anti-CD23 antibodies of interest can be generated in any vector system suitable for expression of functional immunoglobulins.

In addition, the present invention includes gamma-1 or gamma-3 types of human monoclonal antibodies specific for human CD23. Methods of isolating human monoclonal antibodies are well known in the art and include in vitro methods, such as the immune response and in vivo methods of human B cells in tissue culture in vitro, such as in humans in SCID mice. Synthesis of monoclonal antibodies. Preferred methods for preparing human monoclonal antibodies in SCID mice to be introduced into SCID mice after combining in vitro priming of human spleen cells are described in US patent application Ser. No. 08 / 488,376, incorporated herein by reference. This method is advantageous in the case of providing reproducible recovery of monoclonal antibodies with high affinity for the desired antigen, for example a human antigen.

The present invention also encompasses human monoclonal antibodies that bind to CD23 in competition with primate antihuman CD23 monoclonal antibodies 5E8 and 6G5.

Example 1

Generation of Primate Anti-CD23 Antibodies

Five primate monoclonal antibodies specific for CD23 were isolated from macaques substantially in accordance with the method described in US patent application Ser. No. 08 / 379,072 described herein by reference. The exact technique used is described in detail below.

Isolation and Characterization of Anti-human CD23 Monoclonal Antibodies

Purification of Immunogen sCD23 from 8866 Cells

During purification, soluble CD23 (sCD23) was quantified by 3-step ELISA using murine anti-CD23 antibody (the binding site; catalog # MC112) as capture. RPMI 1640 (JR Biosciences; Catalog # 56-509 supplemented with 10% fetal bovine serum (JRH Biosciences) and 4 mM glutamine (JR Biosciences; Catalog # 90114) at 37 ° C. Antigen was partially purified from the culture of 8866 cells maintained in suspension bioreactor. Carbon dioxide was used to maintain pH 7.1. After removal of cells by 0.45 μm filtration, phenylmethyl sulfonyl fluoride (final concentration 0.2 mM, Sigma Chemical Co .; catalog # P-7626) and ethylenediaminetetraacetic acid (final concentration 3 mM, sigma) Chemical Company; Catalog # EDS) was added to the supernatant and the resulting solution was stored at 2-8 ° C. The cell-free supernatant was either hollow fiber ultrafiltration cartridge (A / T Technology; Catalog # UFP-10-C-9A; 10,000 d MWCO) or tangential flow ultrafiltration cartridge (Filtron Corporation; 10,000) at ambient temperature. d MWCO), about 15-20 times concentrated. The concentrated supernatant was sterile filtered and stored at -70 ° C. The thawed concentrate was degreased by addition of SM-2 BioBeads (BioRad Industries; Catalog # 152-3920) at 5 g / L and stirred at 2-8 ° C. overnight. The resin was removed by filtration and the resulting solution was stored at 2-8 ° C. For some preparations of sCD23, the concentrate was fractionated using aluminum sulfate (35-70% (w / v); Fisher; catalog # A702-3) before or after degreasing.

The degreased solution was then purified using affinity chromatography at 2-8 ° C. Affinity matrices were prepared by covalently binding murine anti-CD23 monoclonal antibody (BU38) to Sepharose using CNBr activated Sepharose 4B (Sigma Chemical Company; Catalog # C-9142). Protein A chromatography was used to purify the BU38 antibody from less than 90% homology from ascites (the more binding site; catalog # CUS830). The degreased solution was applied to an affinity column (1.5 × 5 cm) balanced with 1XPBS (Gibco BRL), catalog # 70013-0.32, pH 7.2, and the column was pH 7.2 and 0.05% NP40 (Sigma Chemical). Unbound protein by washing with 1XPBS). Soluble CD23 was eluted using 3.5M MgCl ₂ (Fisher; catalog # 33-500). Fractions containing sCD23 were combined and dialyzed against 1XPBS at 2-8 ° C., pH 7.2 (Baxter Spectra / Por; Catalog # D1615-1). After dialysis, the protein solution was concentrated by centrifugation using a Centriprep 10 spin filter (Amicon Corporation; MWCO 10,000d) and the preparation was stored at -70 ° C. Purity of sCD23 was estimated to be less than 70% as determined using SDS-PAGE analysis (4-20% precast gel, Novex Corporation) and Coomasie staining.

Immune Responses in Primates and Isolation of Immune Cells

Cynomolgus monkeys (White Sands Research Center, Alamogordo, New Mexico) were purified from supernatants of human RPMI 8866 cells [B cell lymphoma, Hassner and Saxon, J. Immunol., 132: 2844 (1984)]. Immunized with soluble CD23. Each monkey was 167 μl of temuritide (co-peptide) (Sigma, St. Louis, Cat. # A 9519) and 333 μl of 3 × Probox (PROVAX; registered) (Idek Pharmaceuticals Corporation) Immunization every three weeks with 200 μg of soluble CD23 in 500 μl PBS mixed with. Immunization was performed intradermal, intraperitoneal, intramuscular and subcutaneous. Appropriate concentrations of anti-CD23 antibodies in monkey serum were measured by ELISA on 8866 cells and compared with blood previously collected from the same monkey.

Sacrifice monkey PRO 978 with a serum titer of 50,000, surgically remove the spleen and lymph nodes, transport to iced Pharmaceuticals on ice, 10% calf fetal serum, 2 mM L-glutamine, 2 mM sodium pyruvate And sterile RPMI-1640 (Kipco Vial, Gaitersberg, MD, catalog # 21870-050) supplemented with 50 μg / ml gentamicin. Upon arrival, the spleen was homogenized by squeezing through a wire mesh with glass pistils. Erythrocytes were digested in storage buffer based on ammonium chloride and the remaining lymphocytes collected and washed at least three times in RPMI-1640. Lymphocytes were similarly homogenized into a single cell suspension, collected and washed at least three times in RPMI-1640.

Preparation of hybridomas

After the last wash, the cells were counted and then the obtained primate cells were mouse-human heterohigh using standard techniques (Refer Boerner et al., J. Immunol., 147: 86 (1991)). Bridoma cell line H6K6 / B5 [Ref .: Carroll et al., J. Immunol. Methods, 89:61 (1986)] and plated into 96 well dishes (175 dishes or 14,700 wells for the spleen and 17 dishes or 1386 wells for lymph nodes) at 300,000 cells per well.

This process involved mixing lymphocytes with the identified fusion partners in a 2: 1 ratio and slowly resuspending the cells in 50% PEG 1500 (Sigma, catalog # P5402) for 1 minute. These cells were then left to stand for 1 minute and then slowly resuspended in excess RPMI-1640. The cells were then left to stand for 15 minutes before the optical spin at 250 × g. Next, 20% calf fetal serum, 2 mM L-glutamine, sodium pyruvate, non-essential amino acids, and 100 μM hypoxanthine, 16 μM thymidine (Charlingermannheim, Germany, # 623091) and 5.8 μM azaserine (Sigma, catalog Cells were resuspended in RMPI-1640 growth medium supplemented with 50 μg / ml gentamycin containing #A 1164) (HTA). HTA is a selection agent that provides the survival of successfully fused fusion cells (primate lymphocytes fused with heterohybridoma fusion partners).

About 65% of the wells showed growth (10,500 wells). These wells were then screened in the presence of antihuman CD23 by 3-step cellular ELISA.

ELISA course

The first step of the ELISA involved transferring 50 μl of supernatant from each well to a 96 well plate previously coated with 10 ⁵ 8866 cells per well (CD23 positive cell line). These plates were prepared by first coating the plates with 50 μl of an aqueous solution containing 20 μg / ml poly L-lysine (Sigma, catalog # P1399, MW 150,000-300,000) for 30 minutes at room temperature. Residual solution was removed (“flicked out”) and the plates were dried. Once dried, 50 μl of 8866 cells in PBS were transferred and spun at 600 g for 5 minutes. 8866 cells were covalently bound to the plate by adding 50 μl of 0.5% glutaraldehyde (Sigma, catalog # G6257) in phosphate buffered saline (PBS) for 15 minutes. Glutaraldehyde was removed (flicked out) and the plates were blocked with 150 μl 100 mM glycine (Sigma, Catalog # G-2879) in 0.1% BSA-PBS. After addition of the supernatant, the plates were incubated for 1 to 2 hours at 37 ° C. and washed 7 to 9 times with tap water and 1% dry creamed milk in PBS-0.05% Tween 20 (Sigma, catalog # P1379) (bones) Goat anti-human IgG coupled to horseradish peroxidase (HRPO) (Southern Biotech, Birmingham, Alabama, catalog # 2040-05) diluted at a ratio of 1: 2000. Antibody was added. Plates were incubated at 37 ° C. for 45 minutes and washed again 7 to 9 times with tap water. The presence of HRPO is detected by color development after adding TMB reagent (Kirkegaad & Perry, Catalog # 50-76-02 and 50-65-02, Gaitersberg, MD) at 100 μl / well It was. The reaction was stopped by adding 25 μl 4N H ₂ SO ₄ . Optical density (OD) was measured at 470 nM on a spectrophotometer (Titertek Multiscan). OD values 2 times larger than the baseline were recorded as positive.

The second step of the ELISA was performed to ensure that the supernatant positively recorded in the first ELISA responded to CD23 but not to some unsuitable antigen. This step was performed by testing the supernatants on SupT1 cells (ERC BioServices Corporation, Catalog # 100), CD23 negative human cell line using the same ELISA procedure. Similarly recorded supernatants in both tests were discarded. These results show that 56 of 10,500 wells with growth show the presence of primate monoclonal antibodies that bind to 8866 cells but do not bind to SupT1 cells at two separate screenings at different times.

The third step of the ELISA was performed to determine whether the supernatant identified according to the first two ELISA steps reacted with soluble CD23. In this third ELISA, 96 well plates were contained in 2 μg / ml BG-6 (Biosource International, catalog # CT-CD23-CF, Camarillo, Calif.), And pH 9.3, 50 mM bicarbonate buffer. , Coated with mouse monoclonal antibody that binds to soluble CD23 but does not block CD23-IgE binding overnight at 4 ° C. After removing the coating buffer, 50 μl of semi-purified soluble CD23 diluted in PBS is added to the plate. And incubate for 2 hours at room temperature The plates were washed 7 to 9 times with tap water, then 50 μl of supernatant was added from selected wells The plates were washed 7 to 9 times with tap water, followed by 0.05% Tween. 50 μl of rabbit antihuman IgG (mouse absorbed) -HRPO (Southern Biotech, catalog # 6145-05) diluted 1: 4000 in 1% dry creamed milk in PBS with 20 was added at 37 ° C. Incubated for 2 hours and, washed 7 times to nine times with tap water and developed with TMB as described above. The wells were recorded with a two-fold greater than the OD values on back to positive.

Twenty-one of the 56 wells bound to sCD23 while bound to 8866 cells in ELISA. These wells were expanded and subcloned at least twice by plating the cells with one cell per three wells. After about three months, five suitable hybridomas were obtained that produced primate monoclonal antibodies against CD23.

Antibody Purification by Protein A Method

In essence, the antibody is purified by centrifugation of the culture supernatant to remove cells and debris. The resulting centrifuged sample is then filtered through a 0.2 μm filter. Next, a Protein A Sepharose Fast Flow column is constructed and equilibrated with PBS (pH 7.4). The supernatant is then loaded into the column at a suitable flow rate (2 ml / min). After loading, the wash column is washed with 10 column volumes of PBS (pH 7.4). The antibody was then eluted from the column with elution buffer (0.2M acetic acid, 0.1M glycine, pH 3.5) at a flow rate of 1 ml / min. Then 1 μl of fractions / tube (2.0 M Tris-HCl pH 10.0) containing 100 μl of Tris is collected. The spectrophotometer reading is then taken at 280 nm. The resulting fractions with high absorbance at 280 nm containing the antibody are then collected and dialyzed overnight against PBS. The product is then sterilized through a 0.22 μm membrane and stored at −20 ° C.

Four of these five primate antihuman CD23 monoclonal antibodies have been shown to inhibit IgE production in an in vitro assay measuring IgE production by IL4-hydrocortisone induced peripheral blood mononuclear cell (PBMC) cultures. These results are shown in FIG. Assay conditions are described below. The fifth primate monoclonal antihuman CD23 antibody B3B11 was inactive in this assay.

IL-4 Stimulated IgE Production by Peripheral Blood Monocytes

As described above, the primate antibodies and their primateization of interest for their ability to inhibit IgE production in an in vitro assay that measures the effect of antibodies on IgE production by IL-4 stimulated peripheral blood mononuclear cells. Evaluated form was evaluated.

Substances for In Vitro IL-4 IgE Assay

Flat bottom cluster plate of 48 wells (Costar, catalog # 3548) (1,500,000 PBMCs per 1 ml for 1 well (48 well plates))

Human recombinant IL-4 (Genzyme, catalog # 2181-01; 10 μg (2.5 × 10 ⁷ units).

Anti-CD23 Mabs:

Murine Mab (MHM6; DAKO.Catalog # M763)

Primate Mabs (no preservatives)

Primateization (no preservatives)

HB 101 Foundation Badge: (Irvine Scientific Catalog # T000)

HB 101 Refill: (Irvine Scientific Catalog # T151)

Fetal Bovine Serum: (FBS; Bio-Whittaker, Catalog # 14-501F)

Dimethylsulfoxide: (DMSO; Fisher Scientific Catalog # D128-500)

Hydrocortisone: (Sigma Catalog # H-0888)

Puromycin: (Sigma Catalog # P-7255)

Cyclohexamide: (Sigma Catalog # C-7698)

Histopaque (registered): (Sigma catalog # H-8889)

Hanks Buffered Salt Solution: (HBSS; Irvine Scientific Catalog # 9232)

1% FBS in HBSS

Concentrated Dulbeccos Phosphate Buffered Saline (10 × DPBS; Bio-Wittaker, Catalog # 17-517Q)

Bath clear disinfectant in DPBS (Fischer, catalog # 13-641-334)

solution:

Puromycin solution: 40 μg / ml in HB101 growth medium

Cyclohexamide solution: 200 μg / ml in HB101 growth medium

Hydrocortisone solution: 0.1M solution in DMSO

Anti-CD23 murine Mab was extensively dialyzed to remove preservatives.

HB101 growth medium

HB101 Basis Medium 500ml

10 ml HB101 supplement

5 ml of mycobacterial filtered distilled water (H ₂ O)

FBS 10ml

0.25 ml hydrocortisone solution (final concentration 5 μM)

In vitro assay method

Buffy coat cells are diluted 1: 4 using HBSS at room temperature. Incubate overnight at room temperature to lyse and isolate plasma components, coagulated platelets and fibrin, and buffy coat cells, and these cells are derived from whole blood.

Then 30 μl of diluted buffy coat is overlaid on 15 μl histo fake in a 50 ml Erlenmeyer flask. These tubes are then centrifuged at 1700 rpm for 20 minutes at room temperature without braking (using IEC 216 swing bucket rotors). The white PBMC layer is then collected using a sterile pipette while taking care not to affect other layers. PBMCs (peripheral blood mononuclear cells) are buffy coats that have been precipitated by partially centrifuging through a histo fake (registration) density gradient to form a visually identifiable white cell layer. These cells are collected by pipette, washed with HBSS and then counted using a hemocytometer. Typically, 300-600 million PBMCs can be recovered from a single 450 ml buffy coat package.

The collected PBMCs are then washed three times in 1% FBS / HBSS. Washed cells are collected by centrifugation for 7 minutes at 1300 rpm at 7 ° C.

The hemocytometer is then used to determine the number of cells collected. Cell concentration is adjusted to be about 3 million cells per ml of HB101 growth medium.

Approximately 1.5 million cells (0.5 ml) are then added to each well of a 48 well plate. In general, five replicated samples are prepared for each experiment. Peripheral wells of each plate are not used as cell samples. Thus, these wells are filled using, for example, 0.05% bath clear / DPBS.

Next, 0.5 ml of HB101 growth medium containing the required amount of IL-4 and Mab is added to the wells. IL-4 used is interleukin 4 in recombinant DNA generated humans. Mab used in the assay is a murine, primate or primatized antibody. Typically, IL-4 is added at a final concentration of 100 U / ml and Mab is added at a final concentration of 0.01-3 μg / ml.

Cells are then incubated for 9-11 days at 37 ° C. in a moisture incubator set at 5% CO ₂ . After incubation, the supernatant is collected and the IgE content is measured.

IgE ELISA

The following list is the materials and solutions used in the IgE ELISA.

Materials and Solutions for IgE ELISA

Sulfuric acid, 4M

Coating Buffer: 10 mM Sodium Bicarbonate Buffer, pH 9.6

Concentrated phosphate buffered saline (10 × PBS) stock solution:

NaH ₂ PO ₄ 26.6gm

Na ₂ HPO ₄ 289gm

NaCl 1064gm

Distilled Water (H ₂ O) 10 L

Blocking Buffer: 10% FBS / PBS

Dilution Buffer: 1% BSA / 0.05% Tween 20 / PBS

Wash buffer: 0.05% Tween 20 / PBS

Unlabeled Goat Antihuman IgE (epsilon chain specific): (Tago Catalog # 4104)

Person IgE of Standard: (More Binding Site Catalog # BP094)

HRP labeled goat antihuman IgE: (ride catalog # AHI0504)

TMB peroxidase substrate: (KPL Catalog # 50-76-02)

Peroxidase Solution B: (KPL Catalog # 50-65-02)

Working substrate solution: A mixture of substrate and solution B in a 1: 1 ratio

Imulon II titer plates (Dynatech Labs Catalog # 011-010-3455)

IgE ELISA Method

Each well of the titer plate is coated with 100 μl of coating buffer containing 2 μg goat anti human IgE.

The coated plate is then incubated overnight at 4 ° C.

After incubation, each well in the plate is washed three times with 200 μl of Tween 20 / PBS. After washing, nonspecific binding sites are blocked with 200 μl blocking buffer / well for 1 hour at 37 ° C.

Then, 100 μl of sample or standard was added to each well; Wells are incubated overnight at 4 ° C. After incubation, the samples are tested with or without dilution. A standard concentration curve is prepared using several IgE dilutions ranging from 0.1 to 50 ng / ml.

After incubation overnight, each plate is washed five times with Tween 20 / PBS.

Next, 100 μl horseradish peroxidase (HRP) labeled goat antihuman IgE diluted 1: 10,000 in dilution buffer is added to each drained well. The plate is then incubated at 37 ° C. for 4 hours.

The plates are then washed five times with Tween 20 / PBS and three times with water.

Next, 100 μl of 3,3 ', 5,5'-tetramethylbenzidine working substrate solution is added to each well. The plates are then incubated for 25 minutes at room temperature and dark. After incubation, 50 μl of 4M sulfuric acid is added to stop the development reaction.

The absorbance at 450 and 540 nm is read simultaneously. The absorbance value at 540 nm is subtracted from the background.

Assay for Kd Measurement of Primate Monoclonal Antihuman CD23 Antibodies

Scatchard Analysis Method

1. How to Use Radioactive Isotopes

Wash IODO-BEADS twice with 100mN phosphate buffer, pH 7.4, using 1 mL buffer per two beads. The beads are then dried on filter paper.

Two beads were then added to a 100 μl ¹²⁵ I solution containing about 1 mCi of I, diluted with 200 μl of phosphate buffer and left at room temperature for 5 minutes.

Antibody (50 μg) is added to the preloaded beads. The reaction time for maximum adoption of radioactivity is 6 minutes.

The reaction is stopped by removing the radiolabeled antibody from the reaction vessel.

Then, to remove excess ¹²⁵ I or ¹²⁵ that is not employed from the radiolabeled antibody solution by performing gel filtration. This is done by passing radiolabeled antibodies on a column consisting of 1.5 mL Sephadex-G25, 1.5 mL DEAE Sephadex-A25, and 0.5 mL Amberlite. Radiolabeled antibodies are eluted in 5 mL total volume at a concentration of about 10 μg / mL. (Elution buffer: 1XPBS containing 10% gelatin, 2% sodium azide and 1% BSA).

2. Optimization Test (Direct Combined Study)

1 μl samples are taken and counted on a gamma counter to determine the specific activity of the 10 μg / mL radiolabeled solution.

Yes:

1 × 10 ⁵ cpm / μl × 1000 μl / 10 μg antibody

1 × 10 ⁵ cpm / μg antibody

1 × 10 ⁴ cpm / ng antibody

Molecular weight of antibody = 75,000 ng / nmole

Specific activity

1 × 10 ⁴ cpm / ng × 75,000ng / nmole = 7.5 × 10 ⁸ cpm / nmole

Block the antigen coated plate (to remove nonspecific binding, eg binding with mB7.1-CHO), and replace the base plate (ie, untransfected CHO) with 200 μL / well of blocking buffer (blocking buffer: 1xPBS containing 10% gelatin, 2% sodium azide, 1% BSA and 10% FBS), and blocked for 1 hour at room temperature.

The one or more plates are then washed ten times by hand, typically using tap water.

Next, 10 μg / ml of radiolabeled antibody (50 μl) is titrated by a 2-fold dilution across the plate using a multichannel pipette. Incubate at room temperature for 1 hour.

One or more plates are washed about 6-7 times with 200 μl / well of wash buffer (wash buffer: 1 × PBS containing 10% gelatin and 2% sodium azide).

The number of radioactivity in each well is then determined by counting the number of wells on the gamma counter.

The optimal radiolabeled antibody concentration is the concentration at which the difference between the specific count and the background count is the largest.

3. Scatchard Analysis of Competitive Tests

10 μg / ml of the radiolabeled solution is diluted to the optimal concentration determined in the direct binding experiment.

Antigen coated and backing plates are blocked with 200 μl / well of blocking buffer for 1 hour at room temperature.

The plate is then washed by hand about 10 times with tap water, for example.

The "cold" (unlabeled radioactive) antibody is then titrated in 2-fold dilution in a separate U-bottom titer. The starting concentration of the "cold" antibody should be at least 100 times greater than the optimal radiolabeled antibody concentration.

Yes:

Optimal radiolabeled concentration: 0.5 μg / mL

“Cold” antibody concentration: 100 μg / mL (Note: a 1: 2 titration in the first well will result in a “cold” antibody concentration of 50 μg / mL).

Then 50 μl / well of optimal radiolabeled antibody is added to the wells containing the “cold” antibody.

100 μl / well of the mixed solution is then transferred to the corresponding wells of the antigen coated plate and incubated for 1 hour at room temperature.

It is also desirable to adjust as follows:

a) direct binding of radiolabeled antibody to antigen coated plates (5 wells),

b) direct binding of the radiolabeled antibody to the base plate (5 wells).

After incubation, the plates are washed with 200 μl / well of buffer, for example about 6-7 times.

The number of radioactivity in each well is then determined by counting the number of wells on the gamma counter.

These calculations are determined by subtracting the background count from the count bound to the antigen coated plate to calculate the specific count in each well tested.

4. Calculations for the Scatchard Analysis

The molarity of bound antibody [B] can be determined as follows:

Example: at 50 μg / mL of “cold antibody”

Combined specific count: 4382 cpm

Bound count in the presence of 50 μg / mLdml “cold” ab: 215 cpm

Difference: 4382cpm-215cpm = 4167 cpm

Specific activity (radiolabelled ab): 5.54 × 10 ⁹ cpm / nmole

4167 ÷ 5.54 × 10 ⁹ cpm / nmole = 7.52 × 10 ^-7 nmole

7.53 × 10 ^-7 nmole ÷ 0.05mL (sample volume)

= 1.50 × 10 ^-5 nmole / mL

= 1.50 × 10 ^-8 nmole / mL

[B] = 1.50 × 10 ⁻¹¹ nmole / mL (M)

The total molarity [T] is determined as follows:

50 μg / mL × 1 μmole / 75,000 μg = 6.67 × 10 ^-4 μmole / mL

= 6.67 × 10 ^-7 μmole / mL (M)

[T] = 66667 × 10 ^-11 mmole / mL (M)

Free antibody [F] was determined as follows:

Molarity of free antibody = total antibody minus bound antibody

[F] = (66667 × 10 ^-11 )-(1.50 × 10 ^-11 )

= 66665.5 x 10 ^-11 mmole / mL (M)

Calculation B / F.

Plot B against B / F on Cricket Graph software.

Activity and Affinity of Anti-human CD23 Antibodies According to the Invention

Four of the five isolated primate antihuman CD23 monoclonal antibodies (B3B11, 2C8, 5E8 and 6G5) were IgE in the above-described in vitro assay by IL-4 hydrocortisone induced peripheral blood mononuclear cell (PBMC) cultures. It has been found to inhibit production. These results are shown in FIG. The fifth primate monoclonal antihuman CD23 antibody 3G12 was inactive in this assay.

Two of the four primate monoclonal antihuman CD23 antibodies (B3B11 and 2C8) found to be inactive in this in vitro assay were commercially available mouse antihuman CD23 antibody MHM6 (Cako A / S, Denmark). A / S) catalog # M763) and was found to compete (top panel in FIG. 2). However, in the repeat assays, these antibodies were not as potent IgE inhibitors as MHM6 (data not shown). In contrast, other primate anti-CD23 monoclonal antibodies (5E8 and 6G5) were found to compete with each other but not with MHM6 (middle and lower panels in FIG. 2). Moreover, primate antihuman CD23 monoclonal antibody 5E8 was found to be an inhibitor of IL-4 induced IgE in vitro assays (see FIGS. 1 and 3).

Measuring Inhibition of IL-4 Induced IgE Production by Modified Hu-SCID-Mouse Model and Human In Vivo Anti-CD23 Antibody for Human IgE Synthesis

A modified hu-PBMC-SCID mouse model was developed to measure the effect of the desired antibody on human IgE production induced in vivo. PBMCs obtained from the two donors were incubated with IL-4 in vitro for 2 days. PBMCs were pooled to reconstruct C.B.-17 SCID mouse groups with and without antibodies. Blood was taken from mice on days 14, 21, 28 and 35 and serum IgG and IgE levels were determined by ELISA. This in vivo model was used to assay primates and two different forms of primatized antibodies to CD23 for their ability to inhibit IgE production.

Several combined immunodeficiency scid / scid (SCID) mice reconstituted with human peripheral blood mononuclear cells (hu-PBMC-SCID), CB-17 [Bosma et al., Nature, 301: 527 (1983) ] Used a modified SCID mouse model because it produces a significant amount of human immunoglobulin (Ig) [Moiser et al., Nature, 335: 256 (1988); Moiser et al., J. Clin. Immunol., 10: 185 (1990); Abedi et al., J. Immunol., 22: 823 (1992); and Mazingue et al., Eur. J. Immunol., 21: 1763 (1991). The predominant isotype of human immunoglobulin (Ig) produced in hu-PBMC-SCID mice is IgG. In general, IgM, IgA and IgE isotypes have been found to be very low or undetectable, except when PBMC is obtained from a donor with a particular autoimmune or allergic disease. It has also been reported that manipulation of hu-PBMC SCID mouse models with specific cytokines can be provided to generate significant levels of non-IgG isotypes, including IgE [Kilchherr et al., Cellular Immunology, 151: 241 (1993); Spiegelberg et al., J. Clin. Investigation, 93: 711 (1994); and Carballido et al., J. Immunol., 155: 4162 (1995). Hu-PBMC SCID was also used to generate antigen specific Ig when the donor was primed against antigen in vivo.

Accordingly, our goal is to provide suitable human IgE to generate a hu-PBMC SCID mouse model that can be used to test therapeutic efficacy for the treatment of IgE related diseases such as allergic diseases, including the anti-CD23 antibody of interest. Focused on establishing

Substances and Methods:

The following materials and methods were used in the hu-PBMC SCID mouse model described below.

SCID Mice: C.B-17 scid / scid immunodeficient mice were obtained from Taconic (C.B.-17 / IcrTac-scidfDF) and bred in an animal facility at Idec Pharmaceuticals. Mice were housed in sterile microbarrier units with sterile bedding. Animal studies were conducted according to the "Guidelines on the Care and Use of Laboratory Animals" as described in the Commission on Care of Laboratory Animal Resources Commission on Life Science-National Research Council. Animals, DHHS Publ. No. (NIH) 86-23, Bethesda, MD, NIH, (1985).

Human PBMCs: PBMCs were isolated from buffy cots obtained from blood banks by centrifugation via Piccol-Hyfake (Histopaque-1077) as recommended by the manufacturer (Sigma Diagnostics Catalog # 1077-1). Lymphocyte preparations were incubated at the gradient boundaries and washed three times in Hanks Balanced Salt Solution (HBSS) (Bio-Witacker Catalog # 10-527F). For each experiment, PBMCs were obtained from two separate donors and incubated separately in vitro. PBMC was supplemented with 1% HB101 lyophilized supplement (Irvine Scientific Catalog # T000 & T151) containing 5% FCS plus 1000 IU / ml IL-4 (Genzyme, Inc. Catalog # 2181-01) and HB- Resuspend in basal medium at 1-3 × 10 ⁶ cells / ml and incubate with 5% CO ₂ at 37 ° C. for 48 hours. After incubation, cells from different buffy cots were cultured and pooled and used to reconstruct SCID mice.

In vivo assay

50-60 × 10 ⁶ lymphocytes in 200-300 μl volume of HBSS were injected intraperitoneally into mouse group (4-5 per group) on day 0. For groups receiving anti-CD23 antibody, on day 0, PBMC was mixed with anti-CD23 antibody (200-400 μg / mouse) and injected twice on day 7 prior to intraperitoneal injection. All mice received intraperitoneally at 5000 IU per mouse of IL-4 between 0 and 5 days. The group not injected with antibody was used as a control. Mouse blood was collected from the tracheal vein and serum was analyzed for IgG and IgE at 14, 21, 28 and 35 days by ELISA.

8 shows that primate antihuman CD23 monoclonal antibody 5E8 is effective at inhibiting IL-4 induced IgE production in a SCID mouse model in vivo.

Cloning and Expression of Primateized Antihuman CD23 Monoclonal Antibodies

For cloning the immunoglobulin variable domains of primates, primates secreting anti-human CD23 monoclonal antibodies 6G5 and 5E8 using the micro-fasttrack mRNA isolation kit (Invitrogen Catalog # K1520-02) according to the method described by the manufacturer Poly A + RNAs were isolated separately from about 2 × 10 ⁶ cells from heterohybridomas.

The first strand of cDNA was synthesized from poly A + RNA using the cDNA Cycle Kit (Invitrogen Catalog # L1310-01) according to a conventional method.

The light and heavy chain variable regions of 6G5 and 5E8 were then isolated by PCR from cDNA using PCR primers selected based on different consensus families of human immunoglobulins. 5 'primers corresponding to the start of the leader sequence of the light and heavy chain variable regions were selected and 3' primers corresponding to the J region were selected. (Specific primers used to PCR amplify the lambda light chain variable domain of 6G5, kappa light chain variable domain of 5E8, and heavy chain variable domains of 6G5 and 5E8 are listed in Table 1-3). PCR was performed according to standard methods (30 cycles of 1 minute at 94 ° C., 1.5 minutes at 54 ° C., and 2 minutes at 72 ° C. in a hot start 100 tube (Kipco Bial Catalog # 10332-013). PCR was used as a template 5 to 80 μl cDNA (from 2 × 10 ⁶ cells), 2 μl 5 nM dNTP, 1 μl Tag polymerase, 5 μl Tag polymerase buffer, 2 μl 5 ′ primer (25 pmol / μl), 3 'Was carried out in 50 μl of reaction solution containing 2 μl of primer (25 pmol / μl) and 36 μl of water. (Tag polymerase and buffer were obtained from Stratagen catalog # 600131 and dNTP was obtained from the Schöllinger Mannheim catalog # 1581295).

A) The composition of plasmids N5LG1 + 6G5 and N5LG4P + 6G5

1) Cloning of the light chain variable domain of primate monoclonal antihuman CD23 antibody 6G5 by PCR

First PCR amplification of the light chain variable region from the cDNA of primate monoclonal antibody 6G5 showed bands consistent with the lambda light chain variable region in situ. These bands appeared in all reactions using three different initial leader sequence primers. However, PCR products obtained using primers 745 (family 2) were considered more specific because of the relatively high intensity of the PCR product bands.

This PCR product was isolated using a Qiaquick gel extraction kit (Qiagen catalog # 28704). Purified PCR fragments were digested with Bgl II and Avr II inhibitory endonucleases and ligated with mammalian expression vector N5LG1 digested with the same inhibitory endonuclease. Then, 20 μl of the ligation mixture containing the purified PCR product from the 50 μl PCR reaction, 2 μl of 100 mg N5LG1 vector, 10 × ligation buffer (NEB Catalog # 202S), 2 μl of T4 ligase (NEB Catalog # 202S) Incubate overnight at &lt; RTI ID = 0.0 &gt;

Mammalian expression vector N5LG1 contains a gene sequence (eg, regulatory sequence, coating sequence) provided for expression of four individual proteins in mammalian cells. These gene sequences are as follows:

(i) partial immunoglobulin light chains with specific inhibitory endonuclease sites and human lambda light chain constant regions for inserting light chain variable regions;

(ii) partial immunoglobulin heavy chains with specific inhibitory endonuclease sites and human gamma 1 chain constant region coding sequences to insert heavy chain variable regions;

(iii) the neomycin phosphorus transferase gene used to select cells into which the plasmid has been incorporated and resistant to the antibiotic zinetisine (Gibco Bial catalog # 10131-1209); And

(iv) murine dihydrofolate reductase gene (DHFR) provided for selection and genomic amplification when cells are cultured in the presence of methotrexate (MTX, Sigma catalog # A-6770) [Reff et al., Blood, 83: 433-445 (1994).

After ligation, the mixture was digested using a Pme I inhibitory endonuclease that degrades the parent N5LG1 plasmid but does not degrade the N5LG1 plasmid ligated to the light chain variable region of 6G5. After digestion, the mixture was transformed with Epicurian coli® XL1-Blue Receptor Cells (Stragen Catalog # 200249).

100 μl of recipient cells were mixed with 10 μl of the ligation mixture above, fixed for 30 minutes and then heated at 45 ° C. for 30 seconds. This mixture was placed on ice for 2 minutes and then 900 μl of pre-warmed SOC at room temperature was added. (SOC is LB broth Gibco BRL catalog # 10855-013 and 0.02M MgCl ₂ , 0.02M MgSO ₄ and 0.02M D-glucose). After incubation at 37 ° C. for 1 h, the mixture was centrifuged at 4000 g for 1 min and 800 μl of supernatant was decanted. The remainder of the mixture was placed on a plate of LB Agar (Kipco Bial Catalog # 12945-044) containing 50 μl / ml Ampicillin (Amp, Gibco Bial Catalog # 13075-015). Plasmid DNA was isolated from individual colonies of E. coli grown on Amp plates using the Wizard® Miniiprep DNA Purification System (Promega Catalog # A7510).

The isolated plasmid DNA was then characterized by degradation to Bgl II and Avr II following agarose gel electrophoresis. 400 bp of ethidium colored DNA bands indicated a potentially successful cloning of the light chain variable regions.

Confirming this is an immunoglobulin light chain variable region and sequencing was performed using the Sequinase 7-deaza-dGTP DNA sequencing kit (USB catalog # 70990) with sequencing primer 607 and GE 108. (See sequencing primers in Table 4).

Second independent PCR amplification of the light chain from the cDNA of primate monoclonal antibody 6G5 was performed using the 5 'primer initial leader sequence of lambda light chain family 2 (primer 745) and 3' J region primer 926. (See primers for PCR of the 6G5 lambda light chain variable regions of Tables 1-3.) Isolated PCR product (see above description) was cloned into TA vector using the original TA cloning kit (Invitrogen Catalog # K2000-01). Isolated miniprep DNA (see above) was digested with EcoR I inhibitory endonuclease followed by agarose gel electrophoresis. The resulting PCR product contained in the TA vector was then sequenced (as previously described) using Sp6 and M13 (-40) forward primers (see sequencing primers in Table 4). The resulting light chain sequence was identical to the light chain sequence from the first PCR. This entire sequence of the light chain variable region of primate monoclonal antihuman CD23 antibody 6G5 is shown below.

Light Chain Variable Regions of Primate Monoclonal Antihuman CD23 Antibody 6G5

2) Cloning of the heavy chain variable region of primate monoclonal antihuman CD23 antibody 6G5 by PCR

First PCR amplification of the heavy chain variable region from the cDNA of primate monoclonal antibody 6G5 was performed using the set of initial leader sequence primers and 3 ′ J region primer GE244 described above. These primers are described in Tables 1-3 below. This reaction produced 350 base PCR products. This 350 base product (purified as described above) was digested with Nhe I and Sal I, ligated with N5LG1 and digested with the same endonuclease during the first PCR amplification. The resulting ligation mixture was transformed into host cells using the same technique to clone the light chain. The plasmid N5LG1 containing 350 base PCR products was then isolated and sequenced (using sequencing primers 266 and 268). (These sequencing primers are shown in Table 4).

Sequencing indicated that the PCR product contained only a portion of the heavy chain variable region and its amino terminus was deleted (sequence started at framework 2, codon 36).

A second independent PCR reaction was performed to amplify and isolate the heavy chain variable region of primate monoclonal antibody 6G5 using 5 'initial leader sequence primer and 3' J 'region primer GE244 for family 1 (MB1503). (These primers are also listed in Tables 1-3). The resulting PCR product was then cloned into N5LG1 using techniques as described above. This sequence was found to be identical to the first PCR product.

Therefore, in order to clone the entire heavy chain variable region of 6G5 containing the invisible 5 'end, a new long 3' primer (MB1533) comprising CDR3 and the backbone 4 region of the 6G5 variable heavy chain was transferred to the family 1 5 'primer (MB1503). Was used for the third independent PCR reaction. (These primers are also described in Tables 1-3.)

After PCR, a large 420 base PCR product was observed on agarose gel. This PCR product was isolated as previously described and cloned into the TA vector. The resulting PCR product contained in the TA vector was then sequenced. Sequencing indicated that the DNA contained the entire heavy chain variable region and the 3 ′ portion was identical to the portion of the previously cloned partial heavy chain variable region from the first two PCR reactions.

A fourth independent PCR amplification was performed using the same primers as the third PCR amplification. From this amplification, the PCR product was isolated and cloned into the TA vector, as previously described. The sequence of the fourth independent PCR product was found to be identical to that obtained in the third PCR amplification. The sequence comprising the heavy chain variable region of primate monoclonal antihuman CD23 antibody 6G5 is described below.

Heavy chain variable region of monoclonal antihuman CD23 antibody 6G5 in primates

Framework 3

CDR 3

Framework 4

3) Construction of Mammalian Expression Vectors

To insert the cloned heavy chain variable domain of 6G5D into the mammalian expression vector, the heavy chain variable domain (obtained by third independent PCR) in the TA vector was digested with NheI and SalI and digested with the same restriction enzyme as above. And cloned into N5LG1 vector already containing light chain variable domain. The resulting mammalian expression vector was called N5LG1 + 6G5.

To structure the N5LG4P + 6G5 vector, the light and heavy chain variable domains were digested with BglI and AvrII and NheI and SalI, respectively, isolated from N5LG1 + 6G5. Mammalian expression vector N5LG4P vector replaces human gamma 1 with human gamma 4 constant region containing a variant of serine to proline in the hinge region to improve pharmacokinetics and increase immunoglobulin selectivity in vivo Same as N5LG1 described above except for the "P" mutation. The light chain variable domains were first cloned into plasmids, and the heavy chain variable domains were cloned into vectors containing the light chain variable domains using the previously described methods. This mammalian expression vector was termed N5LG4P + 6G5.

B. Structures of plasmids N5KG4P + 5E8, N5KG1 + 5E8, N5KG4P + 5E8N- and N5KG1 + 5E8N-

1. Cloning of the Light Chain Variable Domains of Primate Monoclonal Antihuman CD23 Antibodies by PCR

The first PCR reaction of the light chain variable domain from 5E8 cDNA was performed using a set of kappa initial leader sequence primers and 3 ′ J region primer GE204 (see primers for PCR of the kappa light chain variable domain of 5E8 in Tables 1-3). ). 420 base PCR products were obtained. The isolated 420 base PCR product was digested with BglII and BsiWl restriction endonuclease, cloned into a stray expression vector N5KG4P, and sequenced using GE108 and 377 primers (contained in Table 4). Mammalian expression vector N5KG4P is identical to vector N5LG4P except that it contains a human kappa light chain constant region instead of a human lambda light chain constant region. Sequencing of this 420 polynucleotide DNA shows that it contains the entire kappa light chain variable domain.

A second independent PCR of the light chain variable region was performed using 5 'family 1 primer GE201 and 3' primer GE204 (see primers for PCR of kappa light chain variable domain of 5E8 in Tables 1-3). The isolated PCR product was cloned into the TA vector (using the method already described) and sequenced using Sp6 and T7 promoter primers. Sequencing shows that this PCR product is identical to the product obtained by the first PCR. The full sequence of the light chain variable domain of primate monoclonal antihuman CD23 antibody 5E8 is shown below.

Light Chain Variable Regions of Primate Monoclonal Antibody Antihuman CD23 5E8

leader

Mature protein (number is due to Kabat)

Framework 1

CDR 1

Framework 2

CDR 2

Framework 3

CDR 3

Framework 4

2) Cloning of the heavy chain variable domain of primate monoclonal antihuman CD23 antibody 5E8 by PCR

First PCR of the heavy chain variable domain of 5E8 was performed using a set of 5 'initial leader heavy chain sequence primers and 3' primer GE210 (see primers for PCR of heavy chain variable domains of 6G5 and 5E8 in Table 1). A 420 base PCR product appeared in the family 3 primer reaction. The PCR product was purified and then digested with NheI and SalI and cloned into mammalian expression vector N5KG4P vector (as already described). PCR products were sequenced using 268 and 928 primers (see sequencing primers in Table 4).

A second independent PCR of the heavy chain variable domain of 5E8 was performed using family 3 5 'primer GE207 and 3' primer GE210 (see primers for PCR of heavy chain variable domains of 6G5 and 5E8 in Tables 1-3). The isolated PCR product was cloned into a TA vector using the method described above and sequenced using Sp6 and T7 primers. Sequencing showed that the TAC at codon 91 changed to TGC.

To measure intrinsic codons at 91, a third independent PCR was performed with the same primers as the second PCR (see above). PCR products were cloned back into TA vectors and sequenced using Sp6 and T7 primers. The sequence was found to be identical to the heavy chain variable sequence obtained in the first PCR. Thus, the TGC at 91 in the second independent PCR product is clearly the result of the error that occurred during PCR. This entire sequence of the heavy chain variable domain of primate monoclonal antihuman CD23 antibody 5E8 is shown below.

Heavy chain variable region of primate monoclonal antibody antihuman CD23 5E8

leader

Mature protein (number is due to Kabat)

Framework 1

CDR 1

Framework 2

CDR 2

Framework 3

CDR 3

Framework 4

3) Structure of mammalian expression vector

The heavy chain variable domain in N5KG4P was digested with NheI and SalI, purified and cloned into N5KG4P containing the light chain variable domain of 5E8. This plasmid was then digested with restriction endonuclease as described above. This leads to vectors containing the light and heavy chain variable domains of 5E8. This vector was called N5KG4P + 5E8. The heavy and light chain variable domains of N5KG4P + 5E8 were inserted into mammalian expression vector N5KG1 to prepare N5KG1 + 5E8 vectors.

4) Denaturation of Amino Acids in the Heavy Chain Variable Regions of Primate Monoclonal Antibody 5E8 by Site-Specific Variations and Structure of Mammalian Expression Vectors

There is a potential glycosylation site of an immunoglobulin at asparagine codon 75, based on the sequence of the 5E8 heavy chain variable domain. This potential glycosylation site corresponds to a conserved asparagine ligation glycosylation motif with the following tripeptide sequence: (Asp)-(all amino acids except proline)-(serine or threonine). Thus, a glycosylation variant of 5E8 that did not result in glycosylation at this position due to modification of the glycosylation motif was produced by replacing asparagine codon 75 with lysine (many human immunoglobulins were found at this position). Site specific variation is caused by the following method.

The first PCR was performed using N5KG4P + 5E8, 3 'primer (corresponding to codons 71-79) as template, which was coped 75 (AAC changed to AAG), primer MB1654, and 5' at the beginning of the leader sequence. Primer (primer MB1650) (see PCR primers used to prepare glycosyl variants of heavy chain variable region 5E8 of 5E8 described in Table 5).

Second PCR was performed with the same template using 5 'primers containing the same mutations (primer MB1653) and 3' primers from the ends of framework 4 (primer MB1651) (glycos of the heavy chain variable region of 5E8 in Table 5). PCR primers used for the preparation of misfire variations).

These two PCR products were separated and mixed in the same molar ratio. A third independent PCR was performed using a mixture of the first and second PCR products based on the 5 'primer (MB1650) used for the first PCR and the 3' primer (MB 1651) used for the second PCR ( PCR primers used to prepare glycosylation mutations in the heavy chain variable regions of Table 5. The PCR obtained in the third PCR was found to contain the heavy chain variable domain coding region of 5E8, where asparagine 75 was changed to lysine.

The third PCR product was purified, digested with restriction endonucleases SalI and NheI and cloned into N5KG4P containing only the light chain variable domains. PCR products were sequenced to see if they contained variant heavy chain variable domains. This mammalian expression plasmid was named N5KG4P + 5E8N-.

To construct N5KG1 + 5E8N-, both light and heavy chain variable domains from N5KG4P-5E8N- were digested with Bg1 II and BsiW I, and Nhe I and Sal I, respectively. The light chain variable domain was first cloned and then the heavy chain variable domain was inserted into N5KG1, a mammalian expression plasmid, to generate plasmid N5KG1 + 5E8N-.

Table 1

Primer for PCR of the kappa light chain variable domain of 5E8

TABLE 2

Primer for PCR of the Lambda Light Chain Variable Domain of 6G5

TABLE 3

Primers for PCR of heavy chain variable domains from 6G5 and 5E8

Table 4

Sequencing primers

Table 5

PCR primers used to generate glycosylation variants of the heavy chain variable region of 5E8

C) Composition of Gamma-3 Vectors

There are a number of methods for converting murine antibodies to chimeras in which the heavy and light chain constant regions are replaced with human versions, or the CDRs (complementarity determining regions) are almost substituted with their human equivalents. See Biochem. J. 281: 317, 1992; Proc. Nat. Acad. Sci. USA 86: 10029, 1989; Methods Enzymol. 178: 515, 1989; Cancer Res. 51: 181, 191; Biotechniques 7: 360, 1989; J. Immunol. 143: 3589, 1989; Int. J. Cancer 44: 424, 1989; Proc. Nat. Acad. Sci. USA 86: 3833, 1989.

In addition, in the preceding paragraph, the construction of a vector for expressing primatized gamma-1 and gamma-4 antibodies results in amplification of DNA encoding the light and heavy chain variable regions using PCR, and these regions are appropriate human constant regions. It is demonstrated in detail that this can be achieved by cloning into expression vector (s) encoding the domain. Thus, it will be apparent that similar constructs encoding other human constant region domains can be made so long as a suitable mammalian expression vector is also available that contains a cloning site into which the amplified variable region DNA from the primate antibody is inserted. .

It will also be apparent that PCR primers used to amplify variable region DNA can be designed to accommodate a variety of restriction cloning sites, depending on the vector used.

Vectors encoding constant region domains from human IgG3 are available in the art and can be used to construct the primatized antibodies of the invention. For example, Co et al. Constructed chimeric and humanized antibodies using separate vectors expressing human light and heavy chain constant regions and constructed heavy chain vectors for both human gamma-1 and gamma-3. to provide. In order to clone the variable domain regions of the antibodies of interest to these vectors, it is convenient to position the XbaI restriction sites immediately before the constant regions in the J4 and J3 intron segments for the light and heavy chains, respectively. Co et al., 1996, Cancer Res. 56: 1118-1125; Co et al., 1992, J. Immunol. 148: 1149-1154.

Thus, by designing PCR primers that contain XbaI restriction sites, or sites that produce single stranded overhangs identical to XbaI restriction enzymes (ie, NheI, SpeI), variable regions from primate antibodies can be described in the literature, such as The vectors described can be used to bind human gamma-3 constant region domains. Alternatively, linkers or adapters may be used to facilitate cloning.

CDNA versions of the human gamma-3 constant region can be constructed using DNA synthesis techniques and inserted between the NheI and SpeI sites of the N5KG1 vector instead of the human gamma-1 constant region. Depending on which restriction site is most convenient for cloning a particular variable region of interest, there are other gamma-3 vectors known in the art, and any one of them may be used for the gamma-3 antibodies of the present invention. And can be expressed by Parren et al., 1992, J. Immunol. 148 (3): 695-701; Steplewski et al., 1988, Proc. Natl. Acad. Sci. USA 85: 4852-4856; And U.S. Patent No. 4,975,369, which is incorporated herein by reference.

D) Changes in Primateized Antibodies

The invention also encompasses primatized antibodies containing mutations in the constant region, ie substitutions or deletions. Such mutations can be constructed using specific site mutagenesis techniques described above for glycosylation variants that are well known to those skilled in the art.

Mutated antibodies of the invention are designed to produce the desired change in the level of therapeutic efficacy, ie FcR binding. However, it should be understood that by any mutation in the constant region of the antibody, the basic effector function mediated by the gamma-1 or gamma-3 constant region domains is not changed. In addition, other subclasses of gamma antibodies can be changed by mutagenesis so that they have the same effector functions as antibodies containing gamma-1 or gamma-3 domains. Such antibodies are also included in the present invention.

For example, mutations have been identified that characterize regions of the IgG constant region involved in FcR binding and interaction with Clq complement components and increase or decrease binding affinity. As described in US Pat. No. 5,648,260, which is incorporated herein by reference, changing Leu235 to Glu in the human IgG3 constant region disrupts the interaction of the variant with the human Fc gamma R1 receptor. In addition, mutations in adjacent or proximal regions of the hinge binding region (ie, substitution of residues 34, 236 or 237 by Ala) result in at least affinity for the Fc gamma R1 receptor by alterations of residues 234, 235, 236 and 237. It is affected.

Similarly, US Pat. No. 5,348,876, also incorporated herein by reference, discloses Clq binding, ie, complement mediation, by reducing the length of the hinge region of the human IgG3 constant region and changing the amino acid sequence of this region. Cytolysis was shown to be further improved. Indeed, the variant bound Clq better than wild type IgG3, much better than IgG1.

Thus, it is clear from the art that for the treatment of milder medical diseases, mutations can be used to produce therapeutic antibodies that reduce affinity for human Fc receptors. In addition, by identifying mutations that increase effector functional activity, one can induce a stronger version for use in therapy. Manipulation of genetic sequences to alter drug efficacy is contemplated and should be appreciated as falling within the spirit and scope of the present invention.

E) Expression of Primateized Antibodies in Chinese Hamster Ovary Cells

To isolate primatized antibodies, vectors encoding the antibodies were expressed in Chinese hamster ovary cells using the following protocol.

Large-scale plasmid DNA was purified using the Wizard® WIZARD Maxipreps DNA purification system (Promega catalog # A7421). Purified DNA was digested with Ssp I and BspLUll I, precipitated once with ethanol and resuspended in sterile TE.

Purified endonuclease restricted plasmid DNA was then introduced into Chinese hamster ovary (CHO) dihydrofolate reductase minus DG44 cells using electroporation. The electrophoretic technique used is described below.

Approximately 1.6 × 10 ⁸ CHO cells were spun for 1 minute at 1000 RPM in a suitable sterile Corning tube. The medium was separated and the cells were washed with 15 ml of sterile ice cold SBS (sterile sucrose buffer solution 272 mM sucrose, 7 mM sodium phosphate pH 7.4, 1 mM MgCl ₂ ). SBS was isolated and cells were suspended at a concentration of 1 × 10 ⁷ cells / ml using fresh ice cold sterile SBS and left on ice for 15 minutes. The BTX 600 electroporator was operated and pre-adjusted to 230 volts with the maximum voltage lead set to 500 volts / capacitance and resistance. The capacitance was set to 400 microfaraday and the resistance to 13 ohms (setting R1).

The plasmid DNA (4 μg DNA or 2 μg DNA) and 0.4 ml of cells (4 × 10 ⁶ cells) were then placed in a BTX 0.4 ml cuvette (BTX Catalog # 620). The cuvettes were placed in the BTX 600 stand and the cells were shocked by pressing the auto charge and pulse buttons. About 20 individual electrophoresis was performed with each mammalian expression plasmid.

After the impact, the cuvette was left at ambient temperature for 15 minutes. Cells and DNA from each cuvette, HT supplement (100X supplement contains 10 mM sodium hypoxanthine, 1.6 mM thymidine gibco BRL catalog # 11067-014 plus hypoxanthine or thymidine-free CHO-SSFMII Resuspend in 20 ml. Then, the electroporated cuvette was plated in a 96 well plate (200 μl / well) and placed in a 37 ° C. CO ₂ incubator. The medium was initiated by exchanging the medium (G418, Gibco Bial catalog # 10131-019) to which 400 mg / ml of Geneticin® was added, the cells were grown at 37 ° C. and the cell medium was 3 Exchange every 1 to 5. After 16 days, G418 resistant clones in wells and supernatants were assayed for antibody expression by ELISA, after which clones expressing the highest levels were individually propagated. Was purified as described below.

Immunoglobulin ELISA

Plates (Immulon 2, Dynatech Laboratories, Inc. Catalog # 011-010-3455) were prepared using 200 ng of unlabeled goat antihuman IgG and 100 μl / The wells were coated overnight at 4 ° C. This was done using 20 ml of unlabeled goat antihuman IgG / 10 ml coating buffer / plate (Boehringer Mannheim Ab catalog # 605 400). The coating buffer was then removed from the plate and dried with paper towels. Then 100 μl of dilution buffer / well was added.

The antibody solution and standard (100 ng / ml -2.5 ng / ml) were then added directly to 100 ml dilution buffer in duplicate at 100 μl / well. Antibody solutions and standards are contained in dilution buffer. The solution formed was then incubated at 37 ° C. for at least 1 hour.

After incubation, the contents of each plate were removed and the plates washed five times with tap water. The plate was then dried with paper towels.

After the plate was dried, the second antibody was added at 100 μl / well. This second antibody is a 1 / 10,000 dilution or 1 μl Ab / 10 mL dilution, available from Southern Biotechnology Associates, Inc. Catalog # 2060-05 (Southern Biotechnology Associates, Inc. Catalog # 2060-05). Chlorine antihuman lambda-HRPO added to buffer / plate or Chlorine antihuman lambda-HRPO used as a 1 / 20,000 dilution or 1 μl Ab / 20 mL dilution buffer / 2 plate (Southern Biotechnology Associates, Inc. Catalog Available from # 2070-05).

The contents of the antibody and plate were incubated at 37 ° C. for 1 hour. After incubation, the contents of each plate were removed. The plate was washed five times again with tap water and the washed plates were dried. To the dried plate was added HRPO substrate (TMB microwell-2 components) in an amount of 100 μl / well. (5 ml of TMB peroxidase substrate + 5 ml of peroxidase solution B / plate (Kirdgaard and Perry Labs, TMB Microwell Bicomponent Reagent Catalog # 50-76-00)).

The reaction was stopped by adding 100 μl of 2M H ₂ SO ₄ to each well when the weakest standard (2.5 ng / ml) was visualized on the background. The optical density of the wells in the plate was read using a plate reader, for example, a fixed-length molecular Devices Emax precision microplate reader: OD 450 and OPT2 (OD 540).

ELISA buffer

Coating buffer

Sodium Carbonate 0.8g / ℓ

Sodium bicarbonate 1.55g / ℓ

Adjust pH to 9.5 with about 1 mL 1N HCl

Dilution buffer

0.5% skim milk powder (5 g / L) and 0.01% thimerosal (100 mg / L) in PBS

Examples of ELISAs obtained using the assay described above are described below.

Standard OD450 OD450 Average

100ng / ml 0.805 0.876 0.841

50ng / ml 0.395 0.472 0.434

25ng / ml 0.213 0.252 0.233

10ng / ml 0.089 0.105 0.097

5ng / ml 0.054 0.055 0.055

2.5ng / ml 0.031 0.035 0.033

0ng / ml 0.004 0.006 0.005

Standards in Dilution Buffer

Appropriate dilution of Stock AB to give 1 mg / ml (sterile filtered in physiological saline, protein determination by OD).

Yes :

Chimeric monkey / human anti-CD4 (CE9.1) is 4.18 mg / ml.

24 μl of this material into 76 μl dilution buffer is 1 mg / ml.

50 μl of stock Ab into 450 μl dilution buffer (D.B.) is 100 μg / ml.

450 μl D.B. 50 μl of the mixture into 10 μg / ml. *

1.8 ml D.B. 200 μl of the mixture into 1 μg / ml. *

9 ml D.B. 1 ml of the mixture into 100 ng / ml. *

5 ml D.B. 5 ml of the mixture into 50 ng / ml. *

5 ml D.B. 5 ml of the mixture into 25 ng / ml. *

6 ml D.B. 4 ml of the mixture into 10 ng / ml. *

5 ml D.B. 5 ml of the mixture into is 5 ng / ml. *

5 ml D.B. 5 ml of the mixture into 2.5 ng / ml. *

* Is a standard used in ELISA.

Antibody Purification by Protein A

fair

Culture supernatants were centrifuged to remove cells and debris. The centrifuge was filtered through a 0.2 μm filter. Protein A Sepharose Fast Flow Column (Recombinant Protein A Sepharose Fast Flow) (Pharmacia Biotech Catalog # 71-5000-09) was prepared and equilibrated with PBS (pH 7.4).

The supernatant was loaded onto the column at an appropriate flow rate (eg 2 ml / min). After loading, the column was washed with 10 column volumes of PBS (pH 7.4). Antibodies were eluted from the column using elution buffer (0.2M acetic acid, 0.1M glycine, pH 3.5) at a flow rate of 1 ml / min. 1 ml fractions / tubes containing 100 μl of Tris were collected. Spectrophotometer absorbance readings were taken at 280 nm. The antibody fractions were then collected and dialyzed overnight against PBS pH 7.9. The dialysate was then sterilized by filtration through a 0.22 μm membrane and stored at −20 ° C.

Test results

Known primatized® Gamma-4 antihuman CD23 antibodies were purified and assayed for in vitro induced IgE inhibitory activity. The results are included in FIGS. 3 and 3. This was done using a known in vitro IL-4 IgE assay.

These assay results surprisingly suggest that human gamma-4 antihuman CD23 antibodies are not as active as the corresponding primate antihuman CD23 antibodies, ie they do not significantly inhibit in vitro-induced IgE production.

However, because primates 5E8 and p5E8G4P have potential asparagine binding glycosylation sites in the heavy chain variable region, the effects of glycosylation at this site were studied [both of these antibodies contain N-linked oligosaccharides at this site]. (Data not shown)]. Therefore, in order to prevent glycosylation, the addition of carbohydrate was omitted by changing the asparagine in the glycosylation site to lysine. This mutated antibody was named p5E8G4PN-. The assay results demonstrate that the antibody acts equivalently to p5E84GP in the IL-4 assay (see FIG. 3) and also exhibits an equivalent apparent affinity Kd for human CD23 (see FIG. 4). Thus, these results suggest that the difference in IgE inhibition obtained from 5E8 gamma 4 primate® antibodies compared to primate 5E8 antibodies is not due to glycosylation differences.

Three primate antibodies (p5E8G4P, p5E8G4PN-, and pGG5G4P) were expressed as human gamma-1 variants using substantially the same method. All three gamma-1 antihuman CD23 antibodies, denoted p5E8G1, p5E8G1N- and pGG5G1, respectively, were found to be active in the in vitro IL-4 / IgE assay (see FIGS. 3 and 5).

p5E8G1 was found to be statistically more inhibitory than p5E8G4P at concentrations of 0.3 μg / ml (P [T, t] 1 tail + 0.0055) and 3 μg / ml (P [T <t] 1 tail + 0.0019). . In addition, p5E8G1N- was statistically more than p5E8G4PN- at both concentrations of 0.3 μg / ml (P [T <t] 1 tail + 0.0392) and 3 μg / ml (P [T <t] 1 tail + 0.0310). Inhibitory activity (FIG. 3).

Similarly, p6G5G1 completely inhibits induced IgE production at 3 mg / ml, while p6G5G4P is not (this result is shown in FIG. 5).

Thus, these results suggest that the active Fc region, particularly the region of human gamma-1, is important for induced IgE inhibition by anti-human anti-CD23 antibodies. These results will also be important for anti-CD23 antibody-induced IgE inhibition of humans containing human gamma-3 constant regions, since the gamma-3 constant regions bind to the same FcγR receptor to which gamma-1 binds. It also implies that the same result will be obtained.

Example 2

To confirm our assumptions about the inclusion of the Fc effector moiety in IgE inhibition of the anti-human CD23 antibody, a third primate antibody, denoted 2C8 (also proven to inhibit IgE in vitro), was designated F (ab ') Was switched to ₂ . IgE inhibitory activity was determined using the same IL-4 / IgE assay described above.

material

In this example, the following materials were used:

ImmunoPure F (ab ') ₂ Preparation Kit [Pierce Catalog # 44888]

Digestion Buffer: 20 mM Sodium Acetate Buffer (pH 4.5)

0.1 M citric acid (pH 3.0) (adjust pH with NaOH)

0.1% sodium azide in water

Dialysis Tube: 50,000 MW Cutoff [Spectra Por Catalog # 132 128]

Shaking water bath which can be maintained at 37 ℃

Polystyrene Culture Tube (17x100 mm) [Fischer Catalog # 14-956-6B]

BCA Protein Assay [Pierce Catalog # 23224]

Centricon-50 thickener [Amicon catalog # 4225].

Equilibrium of Immobilized Pepsin

0.25 ml of a 50% slurry of immobilized pepsin was added to a 17 × 100 mm test tube (0.125 ml of gel). Then 4 ml of digestion buffer was added. Pepsin was then separated from the buffer using a serum separator. The buffer was then discarded and the wash process repeated with 4 ml of another buffer. Immobilized pepsin was then resuspended in 0.5 ml of digestion buffer.

Preparation of Immobilized Protein A Columns

Protein A AffinityPak® and immunopure binding and elution buffers were kept at room temperature.

Preparation of 2C8 F (ab ') ₂ Fragments

2C8 F (ab ') ₂ fragments were prepared by methods well known in the antibody art. We decided to use a commercial kit, namely the ImmunoPure F (ab ') ₂ Preparation Kit (Pierce Catalog # 44888), using the manufacturer's protocol.

Lyophilized 2C8 antibody was dissolved in 1 ml of digestion buffer (20 mM sodium acetate buffer, pH 4.5). Then 1 ml of antibody containing sample was added to the tube containing immobilized pepsin.

The antibody and immobilized pepsin were incubated for 4 hours (high speed) in a high speed shaker water bath at 37 ° C. while mixing was kept constant during incubation.

The resulting dissolved F (ab ') ₂ and Fc fragments, and undigested IgG, were recovered from the immobilized pepsin gel using a serum separator. The crude digest was decanted into a clean tube.

To improve recovery of F (ab ') ₂ fragments, immobilized pepsin was washed with 1.5 ml of immunopure IgG binding buffer as needed. The wash was then added to the crude digest.

The antibody fragments were then recovered using a Protein A column. This was done by opening the immobilized Protein A column. Care was taken to prevent bubbles from entering the gel. The stock solution (containing 0.02% sodium azide) was discarded.

The immobilized Protein A column was then equilibrated using 20 ml of binding buffer (included in the ImmunoPure kit). The column was then transferred to a 17 × 100 mm test tube contained in a kit (labeled “F (ab ') ₂ ”).

3 ml of crude digest was fed to the column and flowed completely into the gel. The use of affinity packs is preferred as these columns stop flowing automatically when the height reaches the top frit.

The column was washed with 6 ml of binding buffer. Eluates containing F (ab ') ₂ fragments were collected. This eluate also contains small Fc fragments (which are not bound to the Protein A column) that can no longer be bound to Protein A. However, a substantial portion of these is removed by dialysis.

Dialysis was performed by taking the F (ab ') ₂ containing eluate and dialysis the eluate against phosphate buffered saline at pH 7 using a dialysis tube with a molecular weight cutoff of 50,000 to remove small Fc fragment impurities. (Spectra Pur.) Catalog # 132 128].

This produced F (ab ') ₂ with an optical density of 0.707 (6 mL) at 280 nm. After dialysis and concentration by Centricon-50 concentrator (Amicon catalog # 4225), 2C8 F (ab ') ₂ product was assayed for protein content using the BCA protein assay (Pierce catalog # 23224). The protein content was observed to be 3.76 mg / ml.

2C8 F (ab) ₂ ^′ was assayed for IgE inhibitory activity and was found to be substantially unable to inhibit IgE production in the same in vitro assay described above. These results are included in FIG. 6. In fact, F (ab ') ₂ has been shown to counteract the inhibitory action of induced IgE against monoclonal antibody 2C8. These results are shown in FIG.

Example 3

Both primatized gamma 1 and gamma 4P variants of primate monoclonal 6G5 were evaluated for their action on the inhibition of IgE production induced in vivo in the SCID mouse model described above. p5E8G1N has been shown to be effective as primate 5E8 in inhibiting induced IgE (see FIGS. 8 and 9). While neither primate 6G5 nor primatized p6G5G4P was effective in inhibiting in vivo induced IgE, primateized p6G5G1 inhibited induced IgE production (see FIGS. 9 and 10). These results further demonstrate the conclusions of the inventors that the active Fc region is important for the ability of anti-human CD23 antibodies to effectively inhibit IgE production induced.

Proposed mechanism

Although the results of the present invention have demonstrated that the active Fc region is important for the ability of anti-human CD23 antibodies to effectively inhibit IgE production induced, the molecular mechanism has not been elucidated. However, several hypotheses can be made.

First, it can be assumed that the anti-CD23 antibodies of the invention function to elicit similar signal transduction pathways, as is apparently induced by crosslinking the B cell receptor with anti-BCR gamma-1 antibodies. In this case it was hypothesized that down regulation of the antigen receptor signal results from the collocation of Fc gamma RII and surface Ig (B cell receptor) on the same B cells, leading to down regulation of Ig expression. Ambrosia et al., 1995, Science, 268: 293-297; Ono et al., 1997, Cell 90: 293-301. Indeed, CD23 is upregulated at the surface of IgE expressing B cells, allowing the collimation of CD23 and FcγRII-B receptors through the gamma-1 constant region to trigger similar signal transduction pathways.

Alternatively, inhibition of induced IgE expression may occur through cross-linking of surface IgE on B cells at the same time that the Fc region interacts with the appropriate FcγRII receptor on another cell type, ie, killer T cells, and then It may be “instructed” to cause apoptosis in B cells or may induce cell death in some other way (ie, phagocytosis). For example, US Pat. No. 5,543,144, which is incorporated herein by reference, proposes a similar mechanism for inhibiting IgE expressing B cells with anti-IgE antibodies. As described herein, antibodies of specific IgG subclasses, such as mouse IgG2a and human IgG1 and IgG3, can mediate ADCC (antibody dependent cytotoxicity) carried out by certain Fc-containing phagocyte leukocytes. For example, OKT3, a mouse IgG2a monoclonal antibody specific for human T cell surface antigens (this is the first monoclonal antibody product approved by the FDA to be marketed as a therapeutic), is used in patients to provide rapid deletion of blood T cells. And induced an immunosuppressed state (for kidney transplantation). Russell, PS et al., Transpl. Proc. 17: 39-41 (1985). OKT3 can completely delete circulating T cells at a dose of 5 mg / day / patient. The monoclonal antibodies described in US Pat. No. 5,543,144, in particular in the form of mouse gamma 2a antibodies or human gamma-1 or gamma-3 chains containing human or humanized antibodies, induce analogous IgE expressing B cells by the ADCC mechanism in a similar manner. It was proposed to fruition.

Any of the actual molecular mechanisms for inhibiting IgE expression using anti-CD23 antibodies, but it is clear that the Fc region and corresponding effector function of anti-CD23 antibodies are considered important. As described above, because there is no complement in the in vitro assays of the invention, the phenomenon also involves one or more FcγR receptors. As well as identifying the true molecular components, as well as internal molecules for the cells involved in the signal transduction pathway, it will be possible to design other therapeutic agents that can also be used to inhibit induced IgE expression.

Usability

Because of their ability to effectively inhibit IgE production, it can be comprised of a subject anti-human CD23 comprising a human gamma-1 constant domain, and also gamma-3, which are all diseases in which inhibition of IgE production is therapeutically desirable. It is effective in treating it. Such diseases include, for example, allergic diseases, autoimmune diseases and inflammatory diseases.

Examples of specific diseases that can be treated by administration of a subject anti-CD23 human gamma-1 / gamma-3 constant domain containing an antibody are as follows:

Allergic bronchial aspergillosis; Allergic rhinitis and conjunctivitis autoimmune hemolytic anemia; Melanoma cells; Allergic contact dermatitis; Addison's disease; Atopic dermatitis; Alopecia areata; Alopecia areata; Hereditary disease; Anaphylactic purpura; Anaphylactic reactions; Aplastic anemia; Hereditary angioedema; Idiopathic angioedema; Ankylosing spondylitis; Cranial arteritis; Giant cell arteritis; Dagayas arteritis; Temporal arteritis; asthma; Capillary dilatation ataxia; Autoimmune ovarian inflammation; Autoimmune testicles; Autoimmune endocrine gland dysfunction; Beset's disease; Berger's disease; Burger's disease; bronchitis; Bullous fempigus; Chronic mucocutaneous candidiasis; Kaplan syndrome; Myocardial infarction syndrome; Pericardial syndrome; Heartitis; Abdominal sprue; Chagas disease; Shadiak-Higashi syndrome; Chug-strauss syndrome; Nose liver syndrome; Cold coagulation disease; Crest syndrome; Crohn's disease; Cryoglobulinemia; Latent fibrous alveolitis; Herpes dermatitis; Dermatitis; Diabetes mellitus; Diamond-blackplate syndrome; Digejo syndrome; Discoid lupus erythematosus; Eosinophilic fasciitis; Scleritis; Dritma Elytumum Diutinum; Contoured erythema; Polymorphic erythema; Nodular erythema; Familial Mediterranean fever; Pelti's syndrome; Pulmonary fibrosis; Anaphylactic glomerulonephritis; Autoimmune glomerulonephritis; Glomerulonephritis after streptococcal infection; Glomerulonephritis after transplantation; Membranous glomerulonephritis; Goodpaster syndrome; Graft versus host disease; Immune mediated granulocytopenia; Annular granulomas; Allergic granulomatosis; Granulomatous myositis; Grave's disease; Hashimoto's thyroiditis; Hemolytic disease of the newborn; Idiopathic hemochromatosis; Schlein-Henoch purpura; Chronic active and chronic progressive hepatitis; X histiocytosis; Hypereosinophilic syndrome; Idiopathic thrombocytopenia; Jov syndrome; Childhood dermatitis; Childhood rheumatoid arthritis (childhood chronic arthritis); Kawasaki's disease; Keratitis; Dry keratoconjunctivitis; Rendri-Mullang-Barré-Stroll syndrome; Late species; Loupler's syndrome; Lupus; Riel's syndrome; Lyme disease; Lymphoma granulomatosis; Systemic mastocytosis; Hybrid connective tissue disease; Multiple mononeuritis; Muckle-Wells Syndrome; Mucosal skin lymph node syndrome; Multicardiac renal histiocytosis; Multiple sclerosis; Myasthenia gravis; Colony sarcoma; Systemic necrotic vasculitis; Nephrotic syndrome; Redundant syndrome; Fatty stratitis; Paroxysmal cold hemoglobinuria; Yucheon swell; Lupus erythematosus; Frond ileus; Image vulgaris; Pigeon breeder disease; Irritable pneumonia; Nodular polyarteritis; Rheumatoid multiple muscle pain; Polymyositis; Idiopathic polyneuritis; Portuguese familial polyneuropathy; Preeclampsia / eclampsia; Primary biliary cirrhosis; Progressive systemic sclerosis (sclerosis); psoriasis; Joint psoriasis; Alveolar proteinosis; Pulmonary fibrosis, Raynaud's phenomenon / syndrome; Leidel thyroiditis; Reiter's syndrome, recurrent polycrondritis; Rheumatic fever; Rheumatoid arthritis; Sarcoidosis; Scleritis; Curable cholangitis; Serum diseases; Trident syndrome; Sjogren's syndrome; Stevens-Johnson syndrome; Steel bottle; Subacute sclerosing panencephalitis; Sympathetic ophthalmitis; Systemic erythema lupus; Transplant rejection; Ulcerative colitis; Undifferentiated connective tissue disease; Chronic urticaria; Cold urticaria; Uveitis; Alum; Weber-Christian disease; Wegner granulocytopenia; Biscotti-Aldrich Syndrome.

Among these, preferred precursors that can be treated or prevented by the administration of anti-CD23 antibodies include allergic rhinitis and conjunctivitis, atopic dermatitis; eczema; Zob's syndrome, asthma; Allergic diseases; And chronic inflammatory diseases and diseases including CTL chronic lymphocytic leukemia, typically characterized by high levels of membrane CD23 and high circulating levels of sCD23 (Sarfart et al., Blood 71: 94-98 (1988)).

The amount of antibody useful for producing a therapeutic effect can be determined by standard techniques well known to those skilled in the art. Antibodies will generally be provided by standard techniques in pharmaceutically acceptable buffers and may be administered by any preferred route. Because of the efficacy of the presently claimed antibodies and their resistance to humans, it is possible to repeatedly administer these antibodies to combat various diseases or disorders in the human body.

One skilled in the art can determine, by routine experimentation, how routine the non-toxic amounts of antibodies are for affecting allergic and inflammatory diseases. In general, however, an effective dosage will be about 0.05-100 mg / kg of human body weight per day.

Antibodies of the invention can be administered to a human or other animal in accordance with the aforementioned therapeutic methods in an amount sufficient to produce a therapeutic or prophylactic effect. Such antibodies of the invention can be administered to humans or other animals in conventional dosage forms prepared by combining the antibodies of the invention with conventional pharmaceutically acceptable carriers or diluents according to known techniques. Those skilled in the art will appreciate that a pharmaceutically acceptable carrier or diluent is defined by the amount of active ingredient to be formulated, the route of administration and other well known parameters.

The route of administration of the antibodies of the invention can be oral, parenteral, inhalation or topical administration. As used herein, the term “parenteral administration” includes intravenous, intramuscular, subcutaneous, rectal, intravaginal or intraperitoneal administration. Intravenous dosage forms during parenteral administration are generally preferred.

Daily parenteral and oral administration regimens for using the compounds of the invention to prophylactically or therapeutically induce immunosuppression are from about 0.05 to 100 mg, preferably from about 0.5 to 10 mg per kg of body weight per day. Would be common.

Antibodies of the invention can also be administered by inhalation. The term "inhalation" means intranasal and oral inhalation administration. Suitable dosage forms for such administration, such as aerosol formulations or metered dose inhalants, can be prepared by conventional techniques. Preferred dosages of the compounds of the invention to be used will generally be from about 10 mg to about 100 mg.

Antibodies of the invention can also be administered topically. Topical administration means non-systemic administration and includes the external application of an antibody (or fragment thereof) compound of the invention to the epidermis, narrowing and instillation of such antibody into the ear, eye and nose, wherein such antibody Does not enter into the bloodstream. Systemic administration means oral, intravenous, intraperitoneal and intramuscular administration. Of course, the amount of antibody required for a therapeutic or prophylactic action depends on the antibody selected, the nature and condition of the disease to be treated and the animal to be treated, and ultimately at the physician's decision. Suitable topical dosages of the antibodies of the invention will typically be about 1-100 mg / kg body weight per day.

Formulation

The antibody or fragment thereof may be administered alone, but is preferably provided in a pharmaceutical formulation. The active ingredient may comprise from 0.001% to 10% w / w of the formulation weight, for example from 1% to 2%, for topical administration, and may comprise about 10% w / w of the formulation. , Preferably 0.1% to 1% w / w.

Topical formulations of the present invention may comprise the active ingredient together with one or more acceptable carriers, and may also contain other therapeutic ingredients. The carrier must be "acceptable" in terms of compatibility with other components of the formulation and must not be detrimental to such receptors.

Formulations suitable for topical administration are liquids or semi-liquids suitable for penetration through the skin into areas in need of treatment, such as liniments, lotions, creams, ointments, pastes, which are suitable for administration to the eyes, ears and nose. Drops, and the like.

Drops according to the invention may comprise a sterile aqueous solution or an oil solution or suspending agent and are prepared by dissolving the active ingredient in a suitable aqueous solution of fungicides and / or fungicides and / or other suitable preservatives, preferably Surfactants. The resulting solution is filtered and clarified, transferred to an appropriate container and sealed, sterilized for 30 minutes with pressurized steam or 90-100 ° C. The solution is also filtered and sterilized and transferred to the container by aseptic technics. Examples of fungicides and fungicides suitable for inclusion in eye drops include phenyl mercury nitrate or mercury phenyl mercury acetate (0.002%), benzalconium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the preparation of oily solutions include glycerol, dilute alcohol and propylene glycol.

Lotions according to the present invention include those suitable for application to the skin or eyes. Eye lotions may include fungicides and may be prepared similarly to the preparation of eye drops. The lotion or lining agent for application to the skin may include a fast-drying, refreshing agent such as alcohol or acetone and / or a moisturizer such as glycerol or castor oil or peanut oil.

Creams, ointments or pastes according to the invention are semi-solid formulations of the active ingredient for external application. The formulations may be prepared by mixing the active ingredient, either alone or in solution, in final-separated or powdered form or as a suspension in an aqueous or non-aqueous solution, with an oily or non-fatty base, using a suitable apparatus. have. The base may be selected from the group consisting of hydrocarbons such as hard, soft or liquid paraffins, glycerol, beeswax, metal soaps with alcohols such as propylene glycol or macrogels; Mucus; Natural oils such as almonds, corn, peanuts, castor oil or olive oil; Wool fat or derivatives thereof, or fatty acids such as stearic acid or oleic acid, and the like. The formulations can be combined with anionic, cationic or nonionic surfactants such as, for example, sorbitan esters or polyoxyethylene derivatives. Suspending agents such as natural rubber, cellulose derivatives or other ingredients such as silicaceous silicas and lanolin, and the like.

It will be apparent to those skilled in the art that the optimal dosage and the interval between individual dosages in the antibody or fragment thereof of the present invention are determined by the nature and limitation of the disease to be treated, the form, route and site of administration, and the particular animal to be treated. The optimal state can be determined by known techniques. Appropriate treatment is assessed by those skilled in the art. That is, the recovery of the dose of the antibody or fragment thereof of the present invention given in a daily dose for a given number of days is confirmed by one skilled in the art using known therapeutic assays.

Without further elaboration, one skilled in the art can, using the description hereinbefore, fully practice the present invention. Accordingly, the following is merely an example and does not limit the scope of the present invention.

Capsule composition

The pharmaceutical composition of the present invention, which is a capsule, is prepared by filling a standard two-piece hard gelatin capsule with 50 mg of the antibody or fragment thereof, 100 mg of lactose, 32 mg of talc, and 8 mg of magnesium stearate in powder form.

Injectable Parentable Composition

Pharmaceutical compositions according to the invention suitable for injection administration are prepared by stirring 1.5k weight of an antibody or fragment thereof according to the invention in a volume of 10k of propylene glycol and water. The resulting solution is filtered and sterilized.

Ointment Composition

10 g of the antibody or fragment thereof of the present invention.

100.0 g of white soft paraffin.

The antibody or fragment thereof according to the invention is dispersed in a small volume of vehicle to produce a soft, homogeneous product. Fill the dispersion with a collapsible metal tube.

Topical Cream Compositions

1.0 g of the antibody or fragment thereof of the present invention.

Polawax GP 200 20.0 g.

2.0 g of anhydrous lanolin.

2.5 g of white beeswax.

0.1 g of methyl hydroxybenzoate.

Fill with distilled water until the total weight is 100.0g.

The polar wax, beeswax and lanillon are heated to 60 ° C. Methyl hydroxybenzoate is added and homogenized by stirring at high speed. The temperature is then lowered to SOOC. The antibody or fragment thereof according to the invention is added and completely dispersed, and the composition is cooled by slow stirring.

Topical Lotion Composition

1.0 g of the antibody or fragment thereof of the present invention.

Sorbitan monolaurate 0.6 g.

Polysorbate 20 0.6 g.

1.2 g of cetostearyl alcohol.

6.0 g of glycerin.

0.2 g of methylhydroxy benzoate.

Purified water B.P. until the total volume is 100.00 ml. replenishment. (B.P. = British Pharmacopoeia)

The methyl hydroxybenzoate and glycerin are dissolved in 70 ml of water at 75 ° C. The sorbitan monolaurate, polysorbate 20 and cetostearyl are melted together at 75 ° C. and added to the aqueous solution. The resulting emulsion is homogenized, cooled by continuous stirring, and the antibody or fragment thereof according to the invention is added to the residue as a suspension. The entire suspension is stirred until homogenized.

Eye Drop Composition

0.5 g of an antibody or fragment thereof according to the present invention.

0.01 g of methyl hydroxybenzoate.

0.04 g of propyl hydroxybenzoate.

Purified water B.P. replenishment.

The methyl and propyl hydroxybenzoates are dissolved in 70 mL of purified water at 75 ° C., and the resulting solution is cooled. Next, the antibody or fragment thereof according to the present invention is added, and the solution is sterilized by filtration through a membrane filter (0.022 Am pore size) and packaged aseptically in an appropriate sterilization container.

Composition for Administration by Inhalation

10 mg of the antibody or fragment thereof and a lubricant, such as polysorbate 85 or oleic acid 0.2-0.5k, are mixed in a 15-20 ml aerosol container, the mixture being freon, preferably in combination (1,2). Dispersed in propellants such as dichlorotetrafluoroethane) and difluorochloromethane and placed in aerosol containers suitable for intranasal or oral administration. Composition for inhalation administration suitable for aerosol containers in a 15-20 ml dose: the antibody or the fragment of the invention is dissolved in ethanol (6-8 ml) and 0.1-0.2k of a lubricant such as polysorbate or oleic acid is added; It is dispersed in propellants, preferably combinations (1,2 dichlorotetrafluoroethane) and difluoromethane, and placed in aerosol containers suitable for nasal or oral administration.

Parenteral Administration Antibody Composition

Antibodies and pharmaceutical compositions according to the invention are particularly useful for injection, i.e. subcutaneously, intramuscularly or intravenously. The composition for injection administration comprises a solution of the antibody according to the invention or a mixture thereof in a acceptable carrier, preferably a water soluble carrier. Various water soluble carriers can be used, such as water, buffered water, 0.4 k saline (normal saline), 0.3% glycine and the like. The use of normal saline is preferred. These solutions are sterile and free of particulate matter. These solutions can be sterilized by known and known sterilization methods. The composition may comprise a pharmaceutically acceptable adjuvant required to control physiological conditions such as pH adjustment and buffers. In the pharmaceutical composition as described above, the concentration of the antibody or fragment thereof according to the present invention may vary widely. The concentration is primarily selected based on the volume, viscosity, etc. of the solution, depending on the chosen method of administration. In general, a suitable intravenous concentration is 1 to 100 mg per ml.

Thus, the pharmaceutical composition according to the invention for intravenous injection may comprise 10 ml of normal saline containing 40-50 mg of the anti-human CD23 of the invention. Methods of preparing pharmaceutically acceptable compositions are well known and will be apparent to those skilled in the art and are described in detail, for example, in Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania. Reflected for reference.

The antibody of the present invention is lyophilized for storage and reconstituted in a suitable carrier prior to use. The technique is known to be effective with known immunoglobulins, and known lyophilization and reconstitution techniques can be used.

According to the intended result, the pharmaceutical composition according to the present invention may be administered as a prophylactic and / or therapeutic agent. In therapeutic applications, the compositions may be administered in an effective amount for treatment or minimal stopping of the disease and complications for a patient already suffering from the disease. In prophylactic applications, the antibody or its cocktail is administered to a patient who is not yet in a disease state to enhance patient resistance.

Single or multiple administrations of the pharmaceutical compositions may be carried out depending on the dosage level and form selected by the treating physician. In some cases, the anti-CD23 antibody of which the pharmaceutical composition of the invention is the subject should be provided in an effective amount sufficient to treat a patient.

It is also noteworthy that the antibodies according to the invention can be used for the design and synthesis of peptides or non-peptide compounds (analogues) useful in the same treatment as antibodies. Saragovi et al., Science, 253: 792-795. (1991)].

While this specification describes specific embodiments of the invention for purposes of illustration, various modifications may be made without departing from the scope of the invention. Accordingly, the scope of the invention is not limited to the appended claims.

Sequence Listing

(1) General

(i) Applicants: Lev, Mitchell E .; Chloe, William S .; Nakamura, Takehiko

(ii) Name of the Invention: Gamma-1 and Gamma-3 Antihuman CD23 Monoclonal Antibodies and Methods of Using them as Therapeutic Agents

(iii) number of sequences: 35

(iv) Contact Information:

(A) Recipients: Burns, Design, Sweaker & Matisse

(B) Distance: Blood. Five. Box 1404

(C) City: Alexandria

(D) State: Virginia

(E) Country: United States

(F) Zip code: 22313-1404

(v) computer readable form:

(A) Media type: floppy disk

(B) Computer: IBM PC compatible model

(C) working system: PC-DOS / MS-DOS

(D) Software: Patentin Release # 1.0, Version # 1.30

(vi) Current application status:

(A) Application number: US 08 / 803,085

(B) Application date: 1997-2-20

(C) Classification:

(viii) Patent Attorneys / Agents

(A) Statement: Teskin, Robin L.

(B) registration number: 35,030

(C) Reference / Document Number: 012712-353

(ix) Communications

(A) Telephone: (703) 836-6620

(B) Fax: (703) 836-2021

(2) Regarding SEQ ID NO: 1:

(i) the nature of the sequence:

(A) Length of sequence: 390 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(ix) sequence characteristics:

(A) Name / Key: CDS

(B) Presence location: 1..390

(ix) sequence characteristics:

(A) Name / Key: mat_peptide

(B) Presence Location: 58..390

(xi) Sequence description: SEQ ID NO: 1:

(2) Regarding SEQ ID NO 2:

(i) the nature of the sequence:

(A) Length of sequence: 423 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(ix) sequence characteristics:

(A) Name / Key: CDS

(B) Presence location: 1..423

(ix) sequence characteristics:

(A) Name / Key: mat_peptide

(B) Presence location: 58..423

(xi) Sequence description: SEQ ID NO: 2:

(2) for SEQ ID NO 3:

(i) the nature of the sequence:

(A) Length of sequence: 387 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(ix) sequence characteristics:

(A) Name / Key: CDS

(B) Presence location: 1..387

(ix) sequence characteristics:

(A) Name / Key: mat_peptide

(B) Presence location: 67..387

(xi) Sequence description: SEQ ID NO: 3:

(2) Regarding SEQ ID NO 4:

(i) the nature of the sequence:

(A) Sequence length: 411 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(ix) sequence characteristics:

(A) Name / Key: CDS

(B) Presence location: 1..411

(ix) sequence characteristics:

(A) Name / Key: mat_peptide

(B) Presence location: 58..411

(xi) Sequence description: SEQ ID NO: 4:

(2) Regarding SEQ ID NO 5:

(i) the nature of the sequence:

(A) Length of sequence: 41 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 5:

(2) Regarding SEQ ID NO 6:

(i) the nature of the sequence:

(A) Length of sequence: 35 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 6:

(2) for SEQ ID NO 7:

(i) the nature of the sequence:

(A) Length of sequence: 35 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(ix) sequence characteristics:

(A) Name / Key: misc_feature

(B) Presence location: 24

(D) Other information: / week = "nucleotide 24 is N (N = A / G)."

(ix) sequence characteristics:

(A) Name / Key: misc_feature

(B) Presence location: 32

(D) Other information: / week = "nucleotide 32 is N (N = T / G)."

(xi) Sequence description: SEQ ID NO: 7:

(2) Regarding SEQ ID NO: 8:

(i) the nature of the sequence:

(A) Sequence length: 38 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 8:

(2) Regarding SEQ ID NO: 9:

(i) the nature of the sequence:

(A) Sequence length: 29 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(ix) sequence characteristics:

(A) Name / Key: misc_feature

(B) Presence location: 24

(D) Other information: / week = "nucleotide 24 is N (N = C / T)."

(ix) sequence characteristics:

(A) Name / Key: misc_feature

(B) Presence Location: 29

(D) Other information: / week = "nucleotide 29 is N (N = G / C)."

(xi) Sequence description: SEQ ID NO: 9:

(2) Regarding SEQ ID NO: 10:

(i) the nature of the sequence:

(A) Sequence length: 34 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(ix) sequence characteristics:

(A) Name / Key: misc_feature

(B) Presence location: 21

(D) Other information: / week = "nucleotide 21 is N (N = G / A)."

(ix) sequence characteristics:

(A) Name / Key: misc_feature

(B) Presence location: 26

(D) Other information: / week = "nucleotide 26 is N (N = G / C)."

(xi) Sequence description: SEQ ID NO: 10:

(2) Regarding SEQ ID NO 11:

(i) the nature of the sequence:

(A) Sequence length: 34 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(ix) sequence characteristics:

(A) Name / Key: misc_feature

(B) Presence location: 27

(D) Other information: / week = "nucleotide 27 is N (N = A / G)."

(ix) sequence characteristics:

(A) Name / Key: misc_feature

(B) Presence Location: 31

(D) Other information: / week = "nucleotide 31 is N (N = T / C)."

(xi) Sequence description: SEQ ID NO: 11.

(2) Regarding SEQ ID NO: 12:

(i) the nature of the sequence:

(A) Length of sequence: 35 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(ix) sequence characteristics:

(A) Name / Key: misc_feature

(B) Presence location: 23

(D) Other information: / week = "nucleotide 23 is N (N = A / C)."

(ix) sequence characteristics:

(A) Name / Key: misc_feature

(B) Presence Location: 29

(D) Other information: / week = "nucleotide 29 is N (N = T / C)."

(xi) sequence description: SEQ ID NO: 12:

(2) for SEQ ID NO: 13:

(i) the nature of the sequence:

(A) Sequence length: 21 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(xi) Sequence description: SEQ ID NO: 13:

(2) for SEQ ID NO: 14:

(i) the nature of the sequence:

(A) Sequence length: 30 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 14.

(2) Regarding SEQ ID NO: 15:

(i) the nature of the sequence:

(A) Sequence length: 30 base pair amino acids

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 15:

(2) Regarding SEQ ID NO: 16:

(i) the nature of the sequence:

(A) Length of sequence: 33 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) SEQ ID NO: 16:

(2) for SEQ ID NO: 17:

(i) the nature of the sequence:

(A) Length of sequence: 33 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) SEQ ID NO: 17:

(2) Regarding SEQ ID NO: 18:

(i) the nature of the sequence:

(A) Length of sequence: 33 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 18.

(2) Regarding SEQ ID NO: 19:

(i) the nature of the sequence:

(A) Length of sequence: 46 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 19:

(2) Regarding SEQ ID NO: 20:

(i) the nature of the sequence:

(A) Length of sequence: 31 amino acids

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 20:

(2) Regarding SEQ ID NO: 21:

(i) the nature of the sequence:

(A) Sequence length: 51 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 21:

(2) Regarding SEQ ID NO: 22:

(i) the nature of the sequence:

(A) Sequence length: 17 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) sequence description: SEQ ID NO: 22:

(2) Regarding SEQ ID NO: 23:

(i) the nature of the sequence:

(A) Sequence length: 16 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 23:

(2) Regarding SEQ ID NO: 24:

(i) the nature of the sequence:

(A) Sequence length: 20 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 24:

(2) Regarding SEQ ID NO: 25:

(i) the nature of the sequence:

(A) Sequence length: 24 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) sequence description: SEQ ID NO: 25:

(2) Regarding SEQ ID NO: 26:

(i) the nature of the sequence:

(A) Sequence length: 20 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 26:

(2) Regarding SEQ ID NO: 27:

(i) the nature of the sequence:

(A) Sequence length: 21 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 27:

(2) Regarding SEQ ID NO: 28:

(i) the nature of the sequence:

(A) Sequence length: 20 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 28:

(2) Regarding SEQ ID NO: 29:

(i) the nature of the sequence:

(A) Sequence length: 20 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 29:

(2) Regarding SEQ ID NO: 30:

(i) the nature of the sequence:

(A) Sequence length: 20 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) SEQ ID NO: SEQ ID NO: 30:

(2) for SEQ ID NO: 31:

(i) the nature of the sequence:

(A) Sequence length: 20 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 31:

(2) Regarding SEQ ID NO: 32:

(i) the nature of the sequence:

(A) Sequence length: 30 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) sequence description: SEQ ID NO: 32:

(2) Regarding SEQ ID NO: 33:

(i) the nature of the sequence:

(A) Sequence length: 27 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) sequence description: SEQ ID NO: 33:

(2) Regarding SEQ ID NO: 34:

(i) the nature of the sequence:

(A) Sequence length: 27 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 34:

(2) Regarding SEQ ID NO: 35:

(i) the nature of the sequence:

(A) Sequence length: 27 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genomic)

(xi) Sequence description: SEQ ID NO: 35:

Claims (39)

An anti-human CD23 monoclonal antibody comprising a constant region that binds to human Fc gamma receptors and inhibits IgE expression. The anti-human CD23 monoclonal antibody according to claim 1, comprising a primate antigen binding moiety. The antihuman CD23 monoclonal antibody of claim 1, wherein the antibody is a human gamma-1 or human gamma-3 monoclonal antibody. The anti-human CD23 monoclonal antibody according to claim 1, comprising a rodent antigen binding moiety. The anti-human CD23 monoclonal antibody according to claim 1, which is a humanized antibody. The anti-human CD23 monoclonal antibody according to claim 1, which inhibits IgE expression in vitro. 7. The anti-human CD23 monoclonal antibody according to claim 6, which inhibits IL-4 induced IgE expression by B cells in vitro. The antihuman CD23 monoclonal antibody of claim 1, wherein the antibody inhibits IL-4 induced IgE expression in vivo. The anti-human CD23 monoclonal antibody according to claim 1, wherein the binding affinity is 0.01 nM to 1000 nM. The anti-human CD23 monoclonal antibody according to claim 1, wherein the binding affinity of CD23 is 5 nM or more. 10. The anti-human CD23 monoclonal antibody according to claim 9, wherein the CD23 binding affinity is at least 100 nM. The antihuman CD23 monoclonal antibody of claim 1, wherein the variable domain is derived from 5E8, 6G5 or 2C8. The anti-human CD23 monoclonal antibody according to claim 1, wherein the anti-human CD23 monoclonal antibody can inhibit binding of the monoclonal anti-human CD23 antibody 5E8 or 6G5 to CD23. A pharmaceutical composition comprising an anti-human CD23 antibody according to claim 1. A pharmaceutical composition comprising an anti-human CD23 antibody according to claim 2. A pharmaceutical composition comprising an anti-human CD23 antibody according to claim 3. Pharmaceutical composition containing an anti-human CD23 antibody according to claim 4. A pharmaceutical composition comprising an anti-human CD23 antibody according to claim 5. A pharmaceutical composition comprising an anti-human CD23 antibody according to claim 6. A pharmaceutical composition comprising an anti-human CD23 antibody according to claim 7. A pharmaceutical composition comprising an anti-human CD23 antibody according to claim 8. A pharmaceutical composition comprising an anti-human CD23 antibody according to claim 9. A pharmaceutical composition comprising an anti-human CD23 antibody according to claim 10. A pharmaceutical composition comprising an anti-human CD23 antibody according to claim 11. The anti-human CD23 monoclonal antibody according to claim 12, wherein the anti-human CD23 monoclonal antibody can inhibit binding of the monoclonal anti-human CD23 antibody 5E8 to CD23. A method of treating or preventing a disease wherein inhibition of IgE is therapeutically or prophylactically advantageous, comprising administering an effective amount of the antibody of claim 1. A method of treating or preventing a disease wherein inhibition of IgE is therapeutically or prophylactically advantageous, comprising administering an effective amount of the antibody according to claim 2. A method of treating or preventing a disease wherein inhibition of IgE is therapeutically or prophylactically advantageous, comprising administering an effective amount of the antibody according to claim 3. A method of treating or preventing a disease in which inhibition of IgE is therapeutically or prophylactically advantageous, comprising administering an effective amount of the antibody according to claim 4. A method of treating or preventing a disease wherein inhibition of IgE is therapeutically or prophylactically advantageous, comprising administering an effective amount of the antibody according to claim 5. A method of treating or preventing a disease wherein inhibition of IgE is therapeutically or prophylactically advantageous, comprising administering an effective amount of the antibody according to claim 6. A method of treating or preventing a disease wherein inhibition of IgE is therapeutically or prophylactically advantageous, comprising administering an effective amount of the antibody according to claim 7. A method of treating or preventing a disease in which inhibition of IgE is therapeutically or prophylactically advantageous, comprising administering an effective amount of the antibody according to claim 8. A method of treating or preventing a disease wherein inhibition of IgE is therapeutically or prophylactically advantageous, comprising administering an effective amount of the antibody according to claim 12. 30. The method of claim 29, wherein the disease is an allergic disease. The method of claim 29, wherein the disease is an autoimmune disease. The method of claim 29, wherein the disease is an inflammatory disease. 4. The anti-human CD23 monoclonal antibody according to claim 3, which is a human gamma-1 monoclonal antibody. 4. The anti-human CD23 monoclonal antibody according to claim 3, which is a human gamma-3 monoclonal antibody.