JP2003514873A - Treatment of autoimmune diseases with active CD40 binding proteins - Google Patents

Abstract

  (57) [Summary] The present application is a method for treating an autoimmune disease, such as rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus or multiple sclerosis, comprising administering an active CD40 binding protein, such as an anti-CD40 antibody. A method for doing so is disclosed. Preferably, the anti-CD40 antibody is FEK45 or 3 ^ 23.

Description

Detailed Description of the Invention

The present invention relates to the treatment of autoimmune diseases such as rheumatoid arthritis with CD40 binding proteins such as anti-CD40 antibodies.

There is considerable evidence that T lymphocytes drive immune-mediated pathologies, such as autoimmune diseases and transplant rejection, and as a result much effort has been put into these pathologies by controlling the expansion of pathogenic T cells. To devise a successful strategy that can treat
It has been directed in recent years. Increased knowledge about the underlying mechanisms governing T cell activation has led to the de novo generation of compounds that can modulate T cell function and induce a state of immune tolerance that allows for graft receptive and amelioration of autoimmune disease. Enabled the appearance of. For example, CTLA4-Ig is used to block T cell co-stimulation abograti by blocking the use of CD28, one of the essential secondary signals involved in T cell activation.
on ¹ worked well on many models ^2-4 . Furthermore, in humans, CTLA4
Ig has recently been shown to provide very promising results when used in long-term bone marrow allograft acceptance ⁵ . Another surface molecule C formerly called CD40L
In D154, this molecule is mainly CD on type B lymphocytes in dendritic cells (DC) ⁶ .
It has been widely targeted for immunotherapeutic purposes due to its important role it plays in modulating the immune response through the use of 40. Many studies have shown great therapeutic potential for targeting CD154. For example, anti-CD154 monoclonal antibody (mA
b) blocks the induction of autoimmune disorders in a number of animal models ^9-10 and also improves established disease (R-EAE) ¹¹ . Most notably, in vivo
disruption of the CD154-CD40 interaction with the anti-CD154 mAb in o)
It enabled practically unclear renal graft acceptance in non-human primates, thus bringing this approach one step closer to its clinical application. Interestingly, the CD
Inhibitory regulation of 154 expression has an important role even in other well-established therapeutic approaches, such as non-depleting anti-CD4 mAb therapy, thus further highlighting the relevance of CD154 regulation in immune-mediated pathologies. The present inventors have recently observed that. Intuitively, a working anti-CD that mimics the action of CD154
The in vivo use of 40 mAb potently enhances the immune response ¹³ . In vivo administration of a agonizing anti-CD40 mAb may have an important role in promoting a protective immune response against B-cell lymphoma ¹⁵ as well as in promoting antigen-specific immunotherapy against cancer ^15,16. Further recent reports show that. The inventors have also identified the surprising ability of the acting anti-CD40 mAb to control arthritis when administered during disease induction.

Annoying contrasting differences between the widely described in vivo adjuvant effects of agonizing anti-CD40 mAbs and our preliminary findings,
The present inventors have explored the therapeutic potential and possible mechanism of action of an acting anti-CD40 mAb in the CCIA (chronic collagen-induced arthritis) model ¹⁷ . They reported that agonistic anti-CD40 mAbs had a significant therapeutic effect on the development of arthritis, thus indicating their potential clinical use for controlling chronic inflammatory conditions of autoimmune origin. .

A first aspect of the invention provides the use of a working CD40 binding protein, eg an anti-CD40 antibody, in the manufacture of a medicament for treating an autoimmune disease.

The CD40 binding protein can also be a CD40 ligand. Such ligands are known in the art and are shown, for example, in WO 96/26735. Such ligands are expected to behave in a similar manner as the acting anti-CD40 antibody.

A working anti-CD40 antibody may induce activation of CD40. For example, they mimic the action of the natural CD40 ligand by activating CD40.

[0007]   Treatment methods using such binding proteins are also provided.

The term “pharmaceutically effective dose” means an amount of anti-CD40 antibody sufficient to ameliorate an autoimmune disease state in an animal or patient. The required dose can be assessed using routine clinical trials. A typical dose of an antibody is in the range of about 1 μg / kg / day to 10 mg / kg / day of patient or animal body weight. Preferably, this dose is at least 0.01 mg / kg / day.

[0009]   Preferably the animal or patient is a mammal, especially a human.

The anti-CD40 antibody can be introduced into the patient by any method known in the art. For example, the antibody may be delivered intramuscularly, intravenously or parenterally.

The anti-CD40 antibody can be a polyclonal antibody or a monoclonal antibody. Techniques for producing polyclonal or monoclonal antibodies are well known in the art.

As used herein, the term “antibody” refers to an intact molecule capable of binding the CD40 protein and fragments thereof, such as Fa, F (ab ′) ₂ and Fv. The intact CD40 polypeptide or fragment of CD40 can be used as an immunizing antigen. Natural ligands for CD40, such as CD154 (cD40L)
Can be used.

The anti-CD40 antibody can be humanized. That is, leaving the original binding ability,
Amino acids have been replaced in the non-antigen binding regions to better mimic human antibodies. Methods for humanizing antibodies are well known in the art.

Preferably, the antibody is FGK45, or 3 \ 2, when carried out in humans.
3 or a monoclonal antibody having the functional characteristics of soluble CD154.

The autoimmune disease is preferably selected from rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus and diabetes mellitus.

The antibody is preferably administered after initiation of the disease. That is, the antibody is used to treat a disease condition, rather than serve to prevent the disease.

A second aspect of the invention comprises administering a pharmaceutically effective dose of a working anti-CD40 antibody to a sample of an animal, tissue or cell and observing physiological changes to the animal, tissue or cell sample. Methods of testing for autoimmune diseases are provided. Preferably, such methods are performed in vitro on isolated samples of isolated cells, cultured cells or tissues. This affects anti-CD40 on the cells tested
Allows the action of antibodies. Preferably the cell, tissue or antibody is affected, ie it has the characteristics of an autoimmune disease.

[0018]   The antibody and autoimmune disease can be as described above.

Another aspect of the invention is anti-CD40 for use in treating an autoimmune disease.
Providing an antibody. The anti-CD40 antibody and autoimmune disease are as described above.

Yet another aspect of the invention provides an anti-CD40 antibody conjugated to a pharmaceutically effective carrier. Pharmaceutically effective carriers for antibodies are well known in the art.

[0021]   Preferred embodiments of the present invention will be described with reference to the following drawings.

Materials and Methods Mice Chronic Collagen-induced Arthritis Model 36
The R-β TgSWR / J mice TcR-PTG ¹⁷ backcrossed with DBA / 1, after which the transgenic progeny consistently backcrossed against DBA / 1.
Mice from the N10-N15 generation, typified for TcR-β Tg expression, were selected for experiments.

Antibodies The treatment antibodies used were FGK45, anti-mouse CD40 rat IgG ³⁷ (Basel
3 courtesy of Dr. A. Rolink of Institute for Immunology) 3 \ 2
3 (courtesy of Dr. D. Gray of Hammersmith Hospital, London)
, Anti-mouse CD40 rat IgG2a ³⁸ , AFRC MAC-1 (isotype control), anti-dog Chlamydomonas cell wall glycoprotein rat IgG2a (European)
Collection of Animal Cell Culture, Salisbury, UK). The mAb was purified from the culture supernatant by Staphylococcus protein G column (Bioprocessing Durham, UK) and affinity chromatography and sterilized by filter.

The mAbs used for FACS analysis were against mouse CD3 FITC, biotinylated mouse vβ12 TcR (screening), mouse B220PE and streptavidin-PE (all from Pharmingen, San Diego, CA). Finally, the primary antibodies used for immunocytochemistry were anti-mouse B220 biotinylated and isotype control rat IgG2a biotinylated antibodies (both
Pharmingen, San Diego, CA).

The coating / detection antibody pairs used for the ELISA are shown below. IL-2: J
ES6-1A12 / JES6-5H4 (Pharmingen, San Diego, CA), IL-4
: Id11 / BVD6-24G2 (ATCC and Pharmingen, San Di respectively)
ego, CA), IL-5: HB9897 / HB10647 and IFN-γ (R4
-6A2 / XG1.2) (courtesy of Dr. J. Abrams of ATCC, DNAX, Palo Alto, CA).

Preparation of Collagen ³⁹ bovine CII was purified and prepared as previously described. 4 in 0.1 mM acetic acid used for immunization or 0.05 mM Tris-HCl, 0.2 M NaCl, pH 7.4 for in vitro stimulation of spleen cell culture (SPC). Bovine CII was solubilized by stirring overnight at ° C.

Induction and Assessment of Arthritis TcR-β Tg mice (8, 12 weeks old) were immunized with 200 μg of bovine CII (Difco Laboratories, Detroit, MI) emulsified in CFA. The onset of arthritis was assessed daily during the experimental period. The clinical severity of arthritis was graded as follows: 0 = normal, 1 = slight swelling and / or erythema, 2 = significant edematous swelling, 3 = significant edematous swelling + mild joint stiffness, 4 = relaxation. Each paw was graded with a maximum clinical score of 16 for each animal. Hindpaw swelling was recorded with a pair of calipers. All clinical evaluations were conducted blinded.

Immunotherapy Three experimental protocols were performed. In the first set, FGK45, 3 \ 23, A
1 daily dose over 18 days with FRC MAC-1 or PBS
TcR-β Tg mice were treated intraperitoneally from the day of CII in CFA immunization, using four times. The therapeutic approach used a total of 14 intraperitoneal injections over 18 days to administer TcR-β Tg to FGK45,3 \ 23 from the day of disease onset.
, AFRC or PBS intraperitoneally. CII in CFA immunization
Pre-immunization treatment was performed with FGK45, 3 \ 23 or AFRC MAC-1 started 5 days prior to 5 daily ip injections. All antibodies were administered at 300 μg / mouse.

Histological evaluation Hind paws were removed post-mortem, fixed in 10% (w / v) buffered formalin and fixed at 5%.
Decalcified in EDTA. The paws were then embedded in paraffin, sectioned and stained with hematoxylin and eosin or safranin O. Joint changes in the tarsal, metatarsophalangeal, proximal interphalangeal and distal interphalangeal joints can be normal (unaffected), mild (mild synovial hyperplasia), moderate (pannus formation and limited) Erosion) or severe (
Blindly scored as expansive bone and cartilage erosion with loss of joint structure).

Cytokine quantification The spleen was dissected into small pieces to make a single cell suspension, and erythrocyte lysis buffer (Sigm
a) Aldrich, Dorset, UK) to eliminate red blood cells, wash, and wash with 10% (v / v
) Cultured in RPM1 1640 containing heat inactivated FCS, 100 U / ml penicillin, 100 μg / ml streptomycin, 2 × 10 ⁻⁵ M 2-mercaptoethanol and 20 mM L-glutamine. Alone in the medium or 5
With CII of 0 Pg / ml, 72 hours for IL-4 and 24 hours for IL-5 detection, or IFN-gamma detection, 5 × 10 ⁶ cells / ml (200 [mu]
SP was cultured in 96-well plates (Nunc, Uxbridge, UK) at a density of 1 / well). Supernatants were collected, analyzed for cytokines and quantitated by ⁴⁰ sandwich ELISA as previously described.

Serum collagen-specific immunoglobulin quantitation ⁴¹ , Anti-CII antibodies were quantified as previously described ⁴¹ . That is, a microtiter plate (Nunc, Uxbridge, UK) was placed in a Tris buffer (0.05 M Tris, 0).
． 2 μg / ml native bovine C dissolved in 2M NaCl, pH 7.4)
II coated overnight, blocked with 2% BSA and incubated with serially diluted assay sera. Bound IgG was detected by incubation with alkaline phosphatase-conjugated sheep anti-mouse IgG (binding site, Birmingham, UK), followed by incubation with the substrate dinitrophenyl phosphate. Plates were washed at least 3 times with each separate step using 0.001% Tween / PBS. Optical density was measured at 405 nm.

Cytochemistry TcR-β Tg mice were treated from the day of immunization by eight daily ip injections of the designated mAb at 300 μg / mouse.

On day 8 after immunization, the spleen was removed postmortem and the OCT compound (Raymond A. Lamb, Lon.
don, UK) and "quick frozen" in a liquid nitrogen / isopentane bath. Sections were obtained at 5 μm and mounted on microscope slides (Merck Ltd, Leiccestor, UK). Wax-embedded sections were blocked with rat serum and then incubated with biotinylated primary antibody for 1 hour, followed by streptavidin-labeled alkaline phosphatase (Vector, Burlingane, Ca). The localization of the enzyme was visualized using the substrate Fast Red according to the manufacturer's recommendations (DAKO, Carpinteria, Ca) and fixed in 2% formaldehyde for 20 minutes. Finally, slides were washed in water and covered with glycerol gelatin (Sigma Aldrich, Dorset, UK). Slides were washed at least 3 times in PBS at each step.

Statistical Analysis For statistical analysis of data, the Mann-Whitney U test and two-way AN
OVA was applied.

Results Use of CD40 During the Induction Phase of the Autoimmune Response Reduces CCIA Severity In another unpublished study, we found that anti-CD40 mAb-treated mice, as well as anti-CD4 mAb-treated animals, were affected by disease. It was unexpectedly observed to show a significant reduction in severity. To extend our findings, we have used two different acting anti-CD40 mAbs (FGK45 or 3 \ 23), which have previously been used by several groups for the in vivo stimulatory regulation of immune responses. 2 groups of TcR-β
Tg mice were treated from the day of immunization ^14,16,18 . Active anti-CD40 mAb,
AFRC-MAC-1 (300 μg / mouse per day) or PBS was administered 14 times over 18 days from the day of immunization.

FIG. 1 shows one of three separate experiments in which the most severe form of CCIA we observed (onset of disease on days 13-14 as opposed to the usual days 19-20) was observed. 3 shows the inhibitory effect of anti-CD40 mAb treatment on the development of the. Even when administered in these experimental conditions, agonistic anti-CD40 compared to the results obtained from the control treatment group.
The mAbs exerted their ability to control disease onset and significantly reduce disease severity (Figure 1 and Table 1).

[0037]

[Table 1]

Effect of agonizing anti-CD40 mAb treatment on induction of CCIA 8-12 week old sex-matched TcR-βTg mice were challenged with 200 μg / m in CFA.
Immunized with 1 bovine CII. FGK45 (300 μg, n = 10), 3 \ 2
Mice were treated for 18 days with a total of 14 injections of 3 (300 μg, n = 8). The control group consisted of pooled results of mice treated with AFRC-MAC-1 (300 μg, n = 10) or PBS (300 μl, n = 10). Disease progression was monitored daily. a) Date of clinical onset of disease expressed as mean ± SEM of n mice. b) 35 days after immunization, spleens were removed and 5 pooled spleen single cell suspensions were stained with FITC-anti-CD3 and PE-anti-B220 and analyzed by flow cytometry. Ratios were calculated as the number of B220 ⁻ cells over CD3 ⁺ cells. c) Paw swelling was measured for each inflamed hind paw. Results are expressed as the mean swelling of N inflamed paws ± SEM.

[0039]   All results are representative of 3 individually performed experiments.

Consistent with previous report ¹³ , splenomegaly (data not shown) and B lymphocyte expansion (Table 1) were demonstrated in vivo (in v) with either agonistic anti-CD40 mAb.
ivo) Observed in response to administration. Immunohistochemistry in parallel experiments on day 8 post-immunization in 3 \ 23 and FGK45 treated mice also confirmed the expansion of splenic B cell populations. As shown in Figure 2, a clear increase in the number of B220 ^- B cells was observed in both limbic regions of the anti-CD40 mAb (2d and e) treated animals compared to the control groups (2a and B). . Specific cross-reactivity was carefully excluded in each assay group by using the appropriate isotype control mAb (data not shown).

Anti-CD40 mAb Therapy Improves Established CCIA We have previously shown that CCIA animal models are theoretically suitable for conducting therapeutic trials for established autoimmune diseases. ¹⁷ , and it was decided to investigate the effect of agonistic anti-CD40 mAb on the progression of established arthritis.
In three individual experiments, at least 10 daily injections (300 μg / mouse) of FGK45, 3 \ 23, AFRC-MAC-1 or 300 μl / mouse of PBS (the latter two are considered control groups) were administered at least. Five TcR-β Tg mice were treated from the day of clinical onset.

As shown in FIG. 3a, both anti-CD40 mAbs showed a strong inhibitory effect on disease progression, which was revealed within 1 day of the first administration. In fact
Both 3 \ 23 and FGK45 treated mice showed a significant reduction in the clinical severity of arthritis compared to control treated groups (p = 0.017 and p <, respectively).
0.0001). Compared with the results obtained in the control group, FGK45 (p <0.00
01) or 3 \ 23 (p <0.0001) also supported the improvement of arthritis by the significant inhibition of paw swelling measured in mice (Fig. 3b).

Histological Characterization of Anti-CD40 mAb Therapy A blinded histological study was performed to determine if improved disease in anti-CD40 mAb treated mice correlated with reduced joint damage. Histologically, 61, 25% of the joints from the control group were severely damaged by the spread of synovial pannus, fibrin exudates and mononuclear cells accumulating in the synovial cavity (Fig. 4a). As a result, complete loss of bone structure was also observed in half of these severely affected control joints (Fig. 4b). In contrast to the results scored in the control group, only 17.6% of the joints from the 3 \ 23 treated animals were severely damaged, but even in these severe cases the bone structure remained intact. Met. Furthermore, 53% of joints are affected by mild arthritis (Fig. 4c).
), With minimal signs of inflammation and negligible accumulation of inflammatory cells. Finally,
Twelve percent of joints were scored as normal, with no sign of onset of synovitis, thus demonstrating the potent inhibitory effect of this therapeutic approach on the inflammatory process. FG
K45 treatment completely protected 24% of the joints, only 29% were mildly affected, indicating that the cartilage has little erosion (Fig. 4d). The remaining joints showed bone erosion and accumulation of inflammatory cells. Histological analysis of all joints tested is shown in Figure 4e.

Therapeutic administration of anti-CD40 mAb preferentially induces IgG1 CII-specific immunoglobulins Anti-CII antibodies are a trademark of CIA (collagen-induced arthritis) and play a role in the pathogenesis of arthritis. It has been reported to perform ¹⁹ . In CIA, as in other models of autoimmune disease, Th1 autoantigen-specific responses drive the immune response ²⁰ . Thus, for CII in CFA immunizations, high levels of CII-specific IgG2a antibody, Th1-dependent subclass ²¹ , are produced.
The isotype subclass is either Th1 or T predominant in CIA against CII
It is an acceptance index indicating whether the h2 response has been initiated. Therefore, Th1 (IgG
Fluctuations in 2a) or Th2 (IgG1) responses are generally associated with pathogenic Th1 (IgG2)
a) to a potentially protective Th2 (IgG1) response in vivo
o) Monitored in mice after therapeutic manipulations to assess shifts ^22,23 .
Peripheral blood serum CII-specific IgG1 and IgG2a levels were quantified 12 days after disease onset (FIG. 5). Both agonistic anti-CD40 mAbs increased CII-specific IgG2a (Th1) levels by 29 (FGK45) and 72 (3 \ 23) fold compared to control treated mice. Clinically, the levels of IgG1 (Th2) were amplified 46-fold for FGK45 and 260-fold in 3 \ 23 treated mice. These data suggest that anti-CD40 mAb therapy selectively redirects the IgG2a / IgG1 CII-specific antibody ratio, preferring the Th2-regulated IgG1 isotype.

Anti-CD40 mAb Therapy Alters Antigen-Specific T Cell Cytokine Profile The increased anti-CII IgG1 / IgG2a ratio observed after anti-CD40 mAb treatment may be due to anti-CD40 mAb therapy altering T lymphocyte Th1 / Th2 equilibrium. , And thus Th1 (IFN- in different groups of mice
We examined the expression of γ) and Th2 (IL-4 and IL-5) cytokines. CII restimulation of splenocyte cultures (SPC) obtained from mice treated in vivo with 3 \ 23 showed significant suppression of IFN-γ (Fig. 6a).
On the other hand, the level of IL-5 production was increased compared to the pattern observed in isotype control treated mice (Fig. 6c).

A tendency to suppress IFN-γ was observed in SPC from FGK45-treated mice, but no IL-5 production was detected (Fig. 6a, c). Under these experimental conditions, IL-4 was not detected in any of the samples tested (Fig. 6b).

We also tested whether in vitro manipulation with anti-CD40 mAb could affect Th1 / Th2 equilibrium. Under these experimental conditions, CII
And in vitro restimulation of SPC from AFRC-MAC-1 treated mice (control) with 3 \ 23 resulted in a significant reduction in IFN-γ production (Fig. 6d).
Then there was an increase in IL-5 and an initial detection of IL-4 (Figs. 6e and f).
Interestingly, the addition of 3 \ 23 in vitro to SPC from 3 \ 23 treated mice resulted in IL- compared to the values obtained from AFRC-MAC-1 treated SPC in vivo. It was the finding that IFN-γ production was further reduced (FIG. 6d) in parallel with 5 and, more markedly, an increase in IL-4 levels (FIG. 6e, f). SP from AFRC-MAC-1 treated mice with FGK45
An in vitro test of C reduced IFN-γ production (6d) and IL-
5 production was upregulated (6f). These results indicate that agonistic anti-CD40 mAb therapy effectively modifies the pathogenic antigen-specific T cell phenotype, and in the case of 3 \ 23, in particular, in the case of 3 \ 23, an auto-aggressive pro-inflammatory Th1 response and an anti-inflammatory Th2 response. Show that you can turn to.

Pre-immunization Working Agonistic Anti-CD40 Does Not Prevent CCIA It has recently been established that stimulation with a working anti-CD40 mAb can enhance T cell-independent ¹³ and T cell-dependent immune responses ^18. It was Since our data were clearly inconsistent with some well-established studies, we speculated that the crucial factor regarding the type of immune response observed was the timing of anti-CD40 mAb administration. In fact, in studies demonstrating that CD40 stimulation is amplified, agonistic anti-CDm
Abs were introduced ¹⁸ or ¹⁵ before immunization. CII Anti-C before immunization
To determine whether administration of the D40 mAb affects the outcome of the disease, we used FGK45, 3 \ 23, AFRC-M prior to CII in CFA immunization.
Five daily injections (300 μg) of AC-1 or PBS (300 μl / mouse)
/ Mouse). Therefore, we performed a more stringent experimental protocol compared to that used in other pre-immunization experiments ^13,18 . In these experimental conditions, neither FGK45 nor 3 \ 23 prevented CCIA induction (FIG. 7). Therefore, our experimental model shows that the timing of administration of anti-CD40 mAb is decisive for the outcome of its action.

FIG. 8 shows that when the anti-CD40 antibody is IgG1 (TH2) in spleen cells, Ig is selected.
It shows that G2a (TH1 pathogenicity) level is reduced. Cells to AFR
Treatment with Cmac1 isotype monoclonal antibody or anti-CD40 monoclonal antibody. Then, for one month before the assay for IgG2a or IgG1,
The cells were placed in SCID mice.

FIG. 9 shows the effect of anti-CD40 antibody injection in mice on IL-12 production in pathogenic / arthritogenic spleen cells.

Discussion The disruption of the CD40-CD154 axis has recently gained a central place as one of the ideal approaches to control undesired immune responses ⁶ . More interestingly, other therapeutic strategies, such as the use of non-depleting anti-CD4 mAbs, may also be based on the inhibitory regulation of CD154 expression (unpublished results). Clearly, the use of CD40 by acting mAb provide opposite action of a characteristic that is widely used to enhance the immune system when necessary immune response enhancing ^{13-16, 18.} However, the present inventors paradoxically observed that during the course of the initial test with non-depleting anti-CD4 mAb, the acting anti-CD40 mAb could successfully prevent CCIA (unpublished results).

The results described herein further support and extend this surprising finding establishing the therapeutic efficacy of agonistic anti-CD40 mAbs in an autoimmune inflammatory animal model of RA. Most notably, agonizing anti-CD40 mAb therapy appears to be very effective in controlling the progression of CCIA during development. In our experimental model we could detect that a major CII-specific Th2 response was induced in anti-CD40 mAb treated mice, which is one of the mechanisms elicited by agonistic anti-CD40 mAb is autoantigen specific. It is suggested to include the induction of Th2 responses.

One possible explanation for this distinct change in the profile of the Th1 to Th2 T cell response is that it acts as a dominant antigen presenting cell (APC) that drives the expansion and activation of CII-specific T cells. Based on potential changes in cell type. It is well established that antigen presentation by DCs to T cells tends to produce a Th1-type response ²⁵ , especially in the presence of "danger" ²⁴ . On the contrary,
If the non-resting B cells to act as APC, they induce activation of T cells that express IL-4 having a Th2 compatible cytokine profile ^{26-2 8.} The induction of these Th2 responses is well established to offset cell-mediated immune responses ^29-31 . Furthermore, administration of a myelin basic protein (MBP) peptide covalently linked to anti-IgD to enforce the presentation of immunogenic peptides by B cells prevented the induction of cell-mediated autoimmune disease in rats ³² . This prevention was subsequently shown to be due to protective Th2 responses and altered migration characteristics of self-antigen-specific T lymphocytes ³³ . In this situation, agonistic anti-CD40 mAb treated mice show a dramatic expansion of B cells, and when large amounts of CII specific antibodies are present in these mice, the majority of these B cells are CII specific. It is essential to note that Therefore, anti-CD40
The overwhelming population of APCs in mAb-treated mice appears to be non-resting CII-specific B cells. Furthermore, antigen-specific B cells are probably the most efficient APCs in terms of capture capacity, ³⁴ and thus two factors, the large expansion of non-resting B cells and their antigen specificity, work together to produce CII-presenting APCs. It must be remembered to change the balance completely. In this regard, it is also important to note that CD40 use stimulates B cell antigen processing and therefore enhances their APC function ³⁵ . Supporting the hypothesis that operative anti-CD40 mAb engineering during the developing autoimmune chronic inflammatory process may preferably redirect the type of immunological response to the driving self-antigen.

However, mere switching from a Th1 to a Th2-type response would be an anti-CD40m.
It is clear from our study that we cannot explain the full therapeutic effect of Abs. First, the rapid action following injection of a working anti-CD40 mAb is unlikely to be associated only with switching of the dominant APC population. Second, FGK45 and 3 \ 23
The subtle but persistent difference between the actions of s and s shows that alternatives as a yet unclear therapeutic mechanism act in our model. Furthermore, FGK45, a Th2 inducer with apparently minimal potency, shows a more effective therapeutic effect, probably greater than with 3 \ 23. These discrepancies between the two antibodies shown to recognize different epitopes on CD40 (data not shown) indicate that subtle differences between these reagents result in different functional activities.

However, as reported herein, it has been demonstrated to impose a protective immune response against cancer ^14-16 , infectious agent ¹³ , as well as to control the developing inflammatory autoimmune process. The paradox of the dual potency of efficacious anti-CD40 mAbs to enhance the immune response that remains is still inconsistent. We suggest that the timing of administration is the key to explaining these differences and is also the opinion dictated by others ¹⁸ . The inventor has shown that administration of anti-CD40 mAb prior to immunization does not produce the same functional results as compared to after antigenicity testing. Anti-CD
This unique effect of 40 mAb may be a good precursor for the application of anti-CD40 mAb to combat infection or cancer or to treat chronic autoimmune inflammatory processes. In fact, in the first case, the treatment is timed appropriately before or at the time of the antigenicity test in order to support the initiation response, while in the second case the established antigenic cognition is established. Hinder the ongoing process with the intention of deviating.

In summary, agonizing anti-CD40 mAbs successfully regulate chronic autoimmune inflammatory processes, paving the way for their promising use for patients with autoimmune diseases who have not been satisfactorily treated to date. The present inventors have explained the totally unexpected finding that it was.

In cases where an immune response has already occurred, for example, in the immune-inflammatory response in rheumatoid arthritis and other chronic autoimmune diseases, the active anti-CD4
The results demonstrate that 0 antibody is effective in controlling the immune response.

[0058]

[Table 2]

[Brief description of drawings]

FIG. 1. Stimulation of CD40 with agonistic mAbs delays the onset of CCIA and reduces its severity. 8-12 week old sex-matched TcR-βTg mice were treated with 200 μg / m 2 in CFA.
Immunized with 1 bovine Cii. FGK45 (△) (300 μg, n = 10),
3 \ 23 (○) (300 μg, n = 8) was injected 14 times in total to give 1 mouse.
Treated for 8 days. The control group (◆) is AFRC-MAC-1 (300 μg
, N = 10) or PBS (300 μl, n = 10) treated pooled results. Disease progression was monitored daily. Results represent the mean clinical score ± SEM of n mice and are representative of 3 experiments performed separately.

Figure 2: Agonistic anti-CD40 mAb treatment induces B cell expansion. 8-12 week old sex-matched TcR-βTg mice were treated with 200 μg / m 2 in CFA.
Immunized with l bovine type II collagen. 300 μl of PBS (a), 300
μg AFRC-MAC-1 (b), 300 μg 3 \ 23 (c), 300 μg
Mice were treated with daily injections of FGK45 (d) for 8 days from the day of immunization. Spleens removed from animals were snap frozen on day 8 post immunization. 5 μm sections were stained with anti-B220 mAb. The isotype compatible control mAb showed negative staining.

FIG. 3. Treatment of CCIA with a agonizing anti-CD mAb ameliorates established arthritis. 8-12 week old sex-matched TcR-βTg mice were treated with 200 μg / m 2 in CFA.
Immunized with l bovine type II collagen. Mice were treated daily from the day of clinical disease onset. FGK45 (△) (300 μg, n = 10), 3 \ 23 (○)
A total of 10 injections of (300 μg, n = 10) were administered. Control group (◆) is A
FRC-MAC-1 (300 μg, n = 10) or PBS (300 μl, n =
10) Consists of pooled results of mice treated. Clinical score (A) and paw thickness (B) were monitored daily. Results represent the mean ± SEM of n mice and consisted of pooled data from two experiments performed separately.

FIG. 4: Histological evaluation of anti-CD40 mAb therapy Hindfoot arthritis was stained with hematoxylin and eosin and joint erosion was scored as described in Materials and Methods. (A) PBS, (b) AFRC-MAC
-1, Representative joints of mice treated with (c) FGK45 and (d) 3 \ 23.
Results are shown as a percentage of all test paws presented with a specific score (e).

FIG. 5. Therapeutic administration of agonistic anti-CD40 mAb selectively induces CII-specific IgG1 over IgG2a immunoglobulin isotypes. Mice were treated with FGK45 (), 3 \ 23 (□), AFRC-MAC-1 or PBS, the latter two in combination as controls (■), daily for 10 days after disease onset. Peripheral blood serum was collected 12 days after the onset of the disease. CII
Specific IgG2a and IgG1 titers were detected by ELISA. Control CI
Results are expressed as the mean fold increase in CII-specific IgG titers of FGK45 compared to I-specific IgG titers ± SEM.

FIG. 6: Agonistic anti-CD40 mAb therapy alters the cytokine profile of antigen-specific activated T cells. FGK45 () (n = 5), 3 \ 23 (□) (n = 5) or AFRC-M
Individual spleens were collected from AC-1 (■) (n = 5) treated mice (treated as in Figure 3). Single cell suspensions of each individual spleen were treated with 5 × 10 ⁶ cells / ml in the presence of CII (a, b, c) or CII and anti-CD40 mAb (d, e, f) (shown in graph below). Cultured in IL-4 (b, e) and IL-5 (c, f
Samples were analyzed after 24 hours for)) or 72 hours for IFN-γ (a, d). Results represent the mean cytokine expression ± SEM of n individual spleen cell cultures obtained from individual mice ( ^* p <0.05, ^** p <0.001.
). Statistics in graphs d, e and f are comparisons with the values shown in graphs a, b and c, respectively.

FIG. 7. Stimulation of CD40 with a pre-immunizing working mAb does not alter the course of CIA. 8 to 12-week-old sex-matched TcR-βTg mice were treated with FGK45 (Δ) (300).
μg, n = 5), 3 \ 23 (◯) (300 μg, n = 5) for a total of 5 daily injections. The control group (◆) is AFRC-MAC-1 (300 μg, n =
5) or pooled results of mice treated with PBS (300 μl, n = 5). On the last day of treatment, mice were immunized with 200 μg / ml bovine type II collagen in CFA. Disease progression was monitored daily. Results represent the mean clinical score ± SEM of n mice.

FIG. 8: AFRC Ma on anti-CII IgG2a and anti-CII IgG1 production
The action of the c-1 monoclonal antibody and the anti-CD40 antibody is shown.

FIG. 9: I on pathogenic / arthritogenic spleen cells from mice in vivo
The action of anti-CD40 when L-12 is expressed is shown.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 19/02 A61P 25/28 25/28 29/00 101 29/00 101 37/06 37/06 A61K 37 / 02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL , SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL , IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72 ) Inventor Mars, Leonards Theodorus London London W6 8 LH Hammersmith Aspen Lea Road 1 The Matilda and Terence Kennedy Institute of Rheumatology (72) Inventor Ronday, Maru London, England AW 4 2 El Yu Chiswick High Road pre Bend Mansions 3 F-term (reference) 4C084 AA02 BA44 DA01 DA58 NA14 ZA15 ZB08 ZB15 ZC35 ZC41 4C085 AA13 AA14 BB17 CC21 CC31 DD62 EE01 HH17 KA03 KA04 KB82 LL01 LL03

Claims (9)

[Claims] 1. Active C in the manufacture of a medicament for treating an autoimmune disease.
Use of D40 binding protein. 2. A method for treating an autoimmune disease, which comprises administering a pharmaceutically effective dose of a working CD40 binding protein to an animal or a patient. 3. The use or method according to claim 1 or 2, wherein the autoimmune disease is selected from rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus and multiple sclerosis. 4. The method or use according to any one of claims 1 to 3, wherein the active CD40 binding protein is an anti-CD40 antibody. 5. The method or use according to claim 4, wherein the anti-CD40 antibody is a polyclonal antibody or a monoclonal antibody. 6. FGK45,3 \ 2 when said antibody is carried out in humans
The method or use according to claim 5, which is a monoclonal antibody having the functional characteristic of 3 or the functional characteristic of CD154. 7. A method for testing an autoimmune disease, which comprises administering a pharmaceutically effective dose of a working CD40 binding protein to an animal, tissue or cell sample and observing physiological changes to the animal, tissue or cell sample. 8. The method of claim 7, wherein the CD40 binding protein is an anti-CD40 antibody. 9. The method according to claim 7 or 8, wherein the method is carried out in vitro on an isolated specimen of isolated cells, cultured cells or tissues.