WO2001037870A1

WO2001037870A1 - Treatment of autoimmune diseases by an agonistic cd40-binding protein

Info

Publication number: WO2001037870A1
Application number: PCT/GB2000/004511
Authority: WO
Inventors: Marco Londei; Claudia Mauri; Leonaredus Theodorus Mars
Original assignee: Kennedy Rheumatology Inst; Claudia Mauri; Leonaredus Theodorus Mars; Marco Londei
Priority date: 1999-11-25
Filing date: 2000-11-27
Publication date: 2001-05-31
Also published as: JP2003514873A; EP1146902A1; AU1537601A; GB9927757D0

Abstract

The application discloses methods of treating autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosis, diabetes myellitis or multiple sclerosis comprising administering an agonistic CD40 binding protein such as an anti-CD40 antibody. Preferably the anti-CD40 antibody is FGK45 or 3\23.

Description

TREATMENT OF AUTOMMUNE DISEASES BY AN AGONISTIC CD40-BINDING PROTEIN

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

There is compelling evidence that T lymphocytes drive immuno-mediated pathologies, such as autoimmune diseases and transplant rejection, consequentially much effort has been focused in recent years on devising successful strategies able to treat these pathologies by controlling the expansion of pathogenic T cells. The increasing knowledge of the basic mechanisms governing T cell activation has allowed the emergence of a novel generation of compounds able to modulate T cell function and to induce a state of immunological tolerance permitting transplant acceptance and amelioration of autoimmune diseases. For instance the abogration of T cell co-stimulation by blocking CD28 engagement, one of the essential second signals involved in T cell activation¹, with CTLA4-Ig has been successful in many models^2"4. Moreover, in humans, CTLA4Ig has been recently shown to provide very promising results when used in long term bone marrow allograft acceptance⁵. Another surface molecule CD 154, formerly called CD40L, has been extensively targeted for immunotherapeutic purposes due to the key role that this molecule plays in modulating immune responses principally via the engagement of CD40 on B lymphocytes on dendritic cells (DC)⁶. Many studies have indicated the great therapeutic potential of targeting CD 154. For instance anti-CD 154 monoclonal antibody (mAb) prevents the induction of autoimmune disorders in many animal models^7"10, and also ameliorates established diseases (R-EAE)". Most remarkably, disrupting the CD154-CD40 interaction using an anti-CD 154 mAb in vivo, permitted practically indefinite kidney graft acceptance in non-human primates thus moving this approach one step closer to its clinical application¹². Intriguingly, the inventors have recently observed that down-modulation of the CD 154 expression has a key role even in other well established therapeutic approaches, such as non depleting anti-CD4 mAb therapy, thus further stressing the relevance of CD 154 modulation in immuno-mediated pathologies. Intuitively the in vivo use of agonistic anti-CD40 mAbs, mimicking the action of CD154, strongly boosts immune responses¹³. More recent reports have shown that in vivo administration of agonistic anti-CD40 mAb can have a key role in promoting protective immune responses against B cell lymphomas¹⁴, as well as promoting antigen specific immunotherapy for cancer^{15 16}. The inventors have also identified the surprising ability of agonistic anti-C40 mAb to control arthritis when administered during disease induction.

Puzzled by the apparent contrast between the widely described in vivo adjuvant action of agonistic anti-CD40 mAb and the inventor's preliminary findings, the inventors have explored the therapeutic potential and the possible mechanisms of action of agonistic anti-CD40 mAb in a model for CCIA (Chronic Collagen Induced Arthritis)¹⁷. They have reported that agonistic anti-CD40 mAb have a remarkable therapeutic effect on the development of arthritis, thus indicating their potential clinical use to control chronic inflammatory conditions of autoimmune origin.

First aspect of the invention provides use of an agonistic CD40 binding proteins, such as anti-CD40 antibody, in the manufacture of a medicament to treat an autoimmune disease.

The CD40 binding protein may 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 to the agonistic anti-CD40 antibodies.

Agonistic anti-CD40 antibodies are able to induce activation of CD40. For example, they mimic the action of natural CD40 ligands by activating CD40.

Methods of treatment using such binding proteins are also provided.

The term "pharmaceutically effective dose" means an amount of anti-CD40 antibody sufficient to ameriolate the autoimmune diseased state in an animal or patient. The dose required can be assessed using routine clinical trials. The typical dose of antibody will be in the range of about lμg\kg\day to 10 mg\kg\day of patient or animal body weight. Preferably, this dose is at least 0.01 mg\kg\day.

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

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

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

As used herein, the term "antibody" refers to intact molecules as well as fragments thereof, such as Fa,F(ab')₂, and Fv which are capable of binding the CD40 protein. Intact CD40 polypeptide or fragments of CD40 may be used as the immunising antigen. A natural ligand for CD40, such as CD 154 (cD40L) may be used.

The anti-CD40 antibodies may be humanized, that is how amino acids replaced in the non-antigen binding regions in order to more closely resemble a human antibody, whilst still remaining the original binding ability. Methods for humanizing antibodies are well known in the art.

Preferably the antibody is a monoclonal antibody with functional characteristics when implemented in man of FGK45 or 3\23, or soluble CD 154.

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

Antibodies are preferably administered after disease onset. That is, the antibody is used to treat the diseased state, rather than helping to prevent the disease. A second aspect of the invention provides a method of studying an autoimmune disease comprising administering a pharmaceutically effective dose of an agonistic anti-CD40 antibody to an animal, sample of tissue or cell and observing a physiological change to the animal, sample of tissue or cell. Preferably such a method is carried out in vitro on an isolated cell, cultured cell or an isolated sample of tissue. This allows the effect of anti-CD40 antibodies on the cells to be studied. Preferably the cell, tissue, or antibody is diseased, that is it has characteristics of an autoimmune disease.

The antibodies and autoimmune diseases may be as defined above.

Another aspect of the invention provides an anti-CD40 antibody for use to treat an autoimmune disease. The anti-CD40 antibody and autoimmune disease may be as defined above.

Further aspects of the invention provides an anti-CD40 antibody in combination with a pharmaceutically effective carrier. Pharmaceutically effective carriers for antibodies are well known in the art.

Preferred embodiments of the invention will now be described with reference to the following figures:-

Figure 1

Stimulation of CD40, with an agonistic mAb, delays the onset and reduces the severity of CCIA.

Sex -matched TcR-β Tg mice, 8-12 weeks old, were immunised with 200 μg/ml. of bovine Cii in CFA. Mice were treated over an 18 day period with a total of 14 injections of FGK45 (Δ) (300 μg, Λ = 10), 3\23 (O) (300 μg, n = 8). The control group (♦) consists of the pooled results of mice treated with either AFRC-MAC-1 (300 μg, n = 10) or PBS (300 μl, n = 10). Disease evolution was monitored daily. Results represent the mean clinical score of n mice ± SEM and are representative of three individually performed experiments. Figure 2

Agonistic anti-CD40 mAb treatment induces B cell expansion.

Sex-matched TcR-β Tg mice, 8-12 weeks old, were immunised with 200 μg/ml. of bovine collagen type II in CFA. Mice were treated from the day of immunisation for 8 days with daily injections of 300 μl PBS (a), 300 μg AFRC-MAC-1 (b), 300 μg 3\23 (c), 300 μg FGK45 (d). Spleens removed from the animals were snap-frozen 8 days after immunisation, 5 μm sections were stained with anti-B220 mAb. Isotype matched control mAb gave negative staining.

Figure 3

Therapy of CCIA with agonistic anti-CD40 mAb ameliorates established arthritis.

Sex-matched TcR-β Tg mice, 8-12 weeks old, were immunised with 200 μg/ml. of bovine collagen type II in CFA. Mice were treated daily from the day of clinical disease onset, a total of 10 injections of FGK45 (Δ) (300 μg, n = 10), 3\23 (O) (300 μg, n = 10) were administered. The control group (♦) consists of the pooled results of mice treated with either AFRCMAC-1 (300 μg, n = 10) or PBS (300 μl, n = 10). Clinical score (A) and paw thickness (B) were monitored daily. Results represent the mean of n mice ± SEM and consist of the pooled data of two independently performed experiments.

Figure 4

Histological evaluation of anti-CD40 mAb therapy.

Arthritis in the hind paws were stained with haematoxylin and eosin, and the joints erosion was scored as described in Materials and Methods. Joint representative of mouse treated with (a) PBS, (b) AFRC-MAC-1, (c) FGK45 and (d) 3\23. The results are also shown as the percentage of all paws studied that were given the specified score (e). Figure 5

Therapeutic administration of agonistic anti-CD40 mAb preferentially induces CII specific IgGl over IgG2a immunoglobulin isotypes.

Mice were treated daily from disease onset with FGK45 ( ), 3\23 (D), AFRC-MAC-1 or PBS, the latter two combined as control (M), for a total of 10 days. Peripheral blood serum was collected 12 days after disease onset. CII specific IgG2a and IgGl titers were detected by ELISA. The results are represented as the mean fold increase of the CII specific IgG titer of FGK45 and 3\23 relative to the control CII specific IgG titer ± SEM.

Figure 6

Agonistic anti-CD40 mAb therapy alters the cytokine profile of antigen specific activated T cells.

Individual spleens were collected from FGK45 ( )(n = 5), 3\23 (D)(π - 5) or AFRC-MAC-1 (M)(n = 5) treated mice (treatment as described for figure 3). Single cell suspensions of each individual spleen were cultured at 5 x 10⁶ cells/ml. in the presence of CII (a, b, c), or CII and anti-CD40 mAb (d, e, f) (indicated below graph), supernatants were analysed after 24 hrs. for IL-4 (b, e) and IL-5 (c, f)) or after 72 hrs. for IFN-γ (a, d). Results represent the mean cytokine expression of n independent splenocyte cultures obtained from individual mice ± SEM (*p<0.05, **p<0.001). Statistics in graphs d, e and f are relative to the values represented in graphs a, b and c respectively.

Figure 7

Stimulation of CD40, with an agonistic mAb, before immunisation does not alter the course of CIA. Sex-matched TcR-β Tg mice, 8-12 weeks old, were treated with 5 daily injections of FGK45 (Δ)(300 μg, n = 5), 3\23 (O) (300 μg, n = 5). The control group (♦) consists of the pooled results of mice treated with either AFRC-MAC-1 (300 μg, n = 5) or PBS (300 μl n = 5). On the last day of treatment the mice were immunised with 200 μg/ml. of bovine collagen type II in CFA. Disease evolution was monitored daily. Results represent the mean clinical score of n mice ± SEM.

Figure 8 shows the effect of AFRC Mac-1 monoclonal antibodies and anti-CD40 antibodies on anti-CII Ig G2a and anti CII Ig Gl production.

Figure 9 shows the effect of anti-CD40 on IL-12 expression on pathogenic/arthritogenic splenocytes from mice in vivo.

MATERIALS AND METHODS

Mice.

17

The previously described heterozygous TcR-β Tg SWR J mice TcR-β Tg was backcrossed into DBA/1 in order to derive the Chronic Collagen Induced Arthritis model ³⁶, subsequent transgenic offspring was consistently backcrossed against DBA/1. Mice from generation N10-N15 typed for TcR-β Tg expression, were selected for the experiments.

Antibodies.

The treatment antibodies used were FGK45, rat IgG anti-mouse CD40 ³⁷ (kindly provided by Dr A. Rolink, Basel Institute for Immunology); 3\23 (kindly provided by Dr D. Gray,

Hammersmith Hospital, London), rat IgG2a anti-mouse CD40 38 ; AFRC MAC-1 (isotype control), rat IgG2a anti-dog chlamydomonas cell wall glycoprotein (European Collection of Animal Cell Culture, Salisbury, UK). The mAbs were purified from culture supernatants by affinity chromatography, using a staphylococcal protein G column (Bioprocessing, Durham, UK) and filter sterilised.

The mAbs used for FACS -analysis were against mouse CD3 FITC, mouse vβl2 TcR biotinylated (screening), mouse B220 PE, and streptavidin-PE, all obtained from Pharmingen (San Diego, CA). Finally, the primary antibodies used for immunocytochemistry were anti-mouse B220 biotinylated and an isotype control rat IgG2a biotinylated. both obtained from Pharmingen (San Diego, CA).

The coating/detection antibody pairings used for ELISA are as follows, IL-2: JES6- 1A12/JES6-5H4 (Pharmingen, San Diego, CA); IL-4: idl l/BVD6-24G2 (ATCC and Pharmingen, San Diego, CA, respectively); IL-5: HB9897/HB 10647 and IFN-γ (R4- 6A2/XG1.2), ATCC, courtesy of Dr J. Abrams at DNAX (Palo Alto, CA). Preparation of Collagen.

39

Bovine CII was purified and prepared as previously described Bovine CII was solubilized by stirring overnight at 4°C in 0.1 M acetic acid, to be used for immunisation, or 0.05 mM Tris-HCL, 0.2 M NaCl pH 7.4 for in vitro stimulation of splenocyte cultures (SPC).

Induction and assessment of arthritis.

Sex-matched TcR-β Tg mice (8.12 weeks old) were immunised with 200 μg of bovine CII emulsified in CFA (Difco Laboratories, Detroit, MI). The development of arthritis was assessed daily for the duration of the experiment. The clinical severity of arthritis was graded as follows, 0 = normal, 1 - slight swelling and/or erythema, 2 = pronounced oedematous swelling, 3 = pronounced oedematous swelling plus light joint rigidity, 4 = laxity. Each limb was graded, allowing a maximal clinical score of 16 for each animal. Swelling of hind paws was recorded with a pair of callipers. All clinical evaluations were performed in a blinded manner.

Immunotherapy.

Three experimental protocols have been performed. In the first set, TcR-β Tg mice were i.p. treated, from the day of CII in CFA immunisation, with either FGK45, 3\23, AFRC MAC-1 or with PBS, with 14 daily doses over a period of 18 days. In the therapeutic approach, TcR-β Tg were treated i.p. with FGK45, 3\23, AFRC or with PBS from the day of disease onset with a total of 14 i.p. injections over 18 days. The pre-immunisation treatment was performed by 5 daily i.p. injections with FGK45, 3\23 or AFRC MAC-1 initiated 5 days prior to CII in CFA immunization. All antibodies were administered at 300 μg/mouse. Histological evaluation.

Hind paws were removed post mortem and fixed in 10% (w/v) buffered formalin and decalcified in 5% EDTA. The paws were subsequently embedded in paraffin, sectioned and stained with haematoxylin and eosin or safranin O. Arthritic changes in the ankle, the metatarsophalangeal joints, the proximal interphalangeal and the distal interphalangeal joints were scored blindly as normal (unaffected), mild (mild synovial hyperplasia), moderate (pannus formation and limited erosion), or severe (extended bone and cartilage erosion with loss of joint architecture).

Cytokin quantification.

Spleen were teased apart to make a single cells suspension and red blood cells were depleted with a red cell lysis buffer (Sigma Aldrich, Dorset, UK), washed and cultured in RPM1 1640 containing 10% (v/v) heat-inactivated FCS, 100 U/ml. penicillin, 100 μg/ml. streptomycin, 2 x 10"⁵ M 2-mercaptoethanol, and 20 mM L-glutamine. SP were cultured in 96-well plates (Nunc, Uxbridge, UK) at the density of 5 x 10⁶ cells/ml. (200 μl/wells) in medium alone or with 50 μg/ml. of CII for 24 h. for IL-4 and IL-5 detection or for 72 hr. for IFN-γ. Supematants were collected and analysed for cytokines were quantified by a sandwich ELISA as previously described .

Serum collagen specific immunoglobulin quantification.

Anti-CII antibodies were quantified as previously described . Briefly, microtiter plates

(Nunc, Uxbridge, UK) were coated overnight with 2 μg/ml. of native bovine CII dissolved in Tris buffer (0.05 M Tris, 0.2 M NaCl, pH 7.4), blocked with 2% BSA and incubated with serially diluted test sera. Bound IgG was detected by incubation with alkaline phosphatase-conjugated sheep anti-mouse IgG (the binding site, Birmingham, UK) followed by the substrate dinitrophenyl phophate. Plates were washed at each individual step for at least 3 times with 0.001% Tween/PBS. The optical density was measured at 405 nm. Cytochemistry.

TcR-β Tg mice were treated from the day of immunisation with 8 daily intraperitoneal injections of the indicated mAbs at 300 μg/mouse.

Spleens were excised post mortem 8 days after immunisation, embedded in OCT compound (Raymond A. Lamb, London, UK) and 'snap-frozen' in liquid nitrogen/ isopentane bath. Sections were obtained of 5 μm and adhered to microscope slides (Merck Ltd, Leicester, UK). Wax-encircled sections were blocked with rat serum before being incubated for 1 hr. with the biotinylated primary antibodies followed by streptavidin labelled alkaline phosphatase (Vector, Burlingane, Ca). Enzyme localisation was visualised with the substrate fast-red according to the manufacturers' recommendations (DAKO, Carpinteria, Ca) and fixed in 2% formaldehyde for 20'. Finally, slides were washed in water and coverslipped with glycerol gelatin (Sigma Aldrich, Dorset, UK). Slides were washed at least 3 times in PBS at each step.

Statistical analysis.

For the statistical analysis of the data, the Mann- Whitney U test and the 2-way ANOVA were applied.

RESULTS

Engagement of CD40 during the induction phase of the autoimmune response reduces the severity of CCIA.

In another, unpublished, study the inventors unexpectedly observed that anti-CD40 mAb treated mice, like the anti-CD4 mAb treated animals, showed a significant reduction in disease severity. To expand our findings, two groups of TcR-β Tg mice were treated, from the day of immunisation, with two different agonistic anti-CD40 mAbs (FGK45 or 3\23) which have been previously used by several groups for in vivo upregulation of immune responses ¹⁴' ¹⁶' ¹⁸. Agonistic anti-CD40 mAb, AFRC- MAC-1, (300 μg/mouse daily) or

PBS were administrated 14 times over an 18 day period from the day of immunisation.

Figure 1 shows the suppressive effects of the anti-CD40 mAb treatment on the evolution of the most severe (onset of the disease 13-14 as opposed to the usual day 19-20) form of CCIA, which we have observed in 1 out of 3 independent experiments. Even when administered in these experimental conditions both agonistic anti-CD40 mAb exerted their ability to control onset and to significantly reduce the severity of disease when compared with the results obtained from the control treated group (Figure 1 and Table 1).

Table 1.

Effects of agonistic anti-CD40 mAb treatment on the induction of CCIA.

Sex-matched TcR-β Tg mice, 8-12 weeks old, were immunised with 200 μg/ml. of bovine collagen type II in CFA. Mice were treated over an 18 day period with a total of 14 injections of FGK45 (300 μg, n = 10), 3\23 (300 μg, n = 8). The control group consists of the pooled results of mice treated with either AFRC-MAC-1 (300 μg, n - 10) or PBS (n = 10). Disease evolution was monitored daily.

a) Day of clinical onset of disease expressed as the mean of n mice ± SEM.

b) Spleens were removed on day 35 after immunisation, single cell suspensions of 5 pooled spleens were stained with FITC-anti-CD3 and PE-anti-B220 and analysed by flow cytometry. Ratio was calculated as number of B220⁺ cells over CD3⁺ cells.

c) Paw swelling was measured for each inflamed hind paw. Results expressed as mean swelling of n inflamed paws ± SEM.

All results are representative of three individually performed experiments.

In agreement with previous reports 13 splenomegaly (data not shown) and an expansion of

B lymphocytes (Table 1) were observed in response to in vivo administration of either agonistic anti-CD40 mAbs. The increase of the splenic B cell population was also confirmed, by immuno-histochemistry, in a parallel experiment 8 days after immunisation in the 3\23 and FGK45 treated mice. As shown in Figure 2, a clear increase in number of B220⁺ B cells was observed in the marginal zone of both anti-CD40 mAbs (2 d and e) treated animals compared to control group (2 a and B). Aspecific crossreactions were carefully excluded in each analysed sample by using an appropriate isotype control mAb (data not shown).

Anti-CD40 mAb therapy ameliorates established CCIA.

We have previously shown that the CCIA animal model is ideally suited to perform therapeutic studies on established autoimmune disease , and we decided to investigate the effects of agonistic anti-CD40 mAb on the progression of established arthritis. In three independent experiments, at least 5 TcR-β Tg mice were treated from the day of clinical onset with 10 daily injections (300 μg/mouse) of FGK45, 3\23, AFRC-MAC-1 or 300 μl/mouse of PBS, the latter two refereed as control groups.

As shown in Figure 3 a both anti-CD40 mAbs showed a strong suppressive effect on the progression of disease, which was apparent within one day of the first administration. In fact, both 3\23 and FGK45 treated mice manifested a significantly reduced clinical severity of arthritis (p = 0.0017 and p<0.0001, respectively) compared to the control treated group. The amelioration of arthritis was also supported by the significant inhibition of paw swelling measured in mice treated with either FGK45 (pO.OOOl) or 3\23 (p<0.0001) compared to the results obtained in the control group (Figure 3b).

Histological features of anti-CD40 mAb therapy.

Blinded histological examination was carried out to determine whether the amelioration of disease in anti-CD40 mAb treated mice correlated with reduced joints damage. Histologically 61, 25% of the joints from the control group were severely damaged by the expansion of the synovial pannus, fibrin essudate and mononuclear cells, which accumulate in the synovial space (Figure 4a). Consequently, a complete loss of the bone architecture was also observed in half of these severely affected control joints (Figure 4b). In contrast to the results scored in the control group, only 17.6% of joints from 3\23 treated animals were severely damaged, but even in these severe cases the bone architecture was left intact. Moreover, 53% of joints were affected by a very mild arthritis (Figure 4c) with minimal signs of inflammation and a negligible accumulation of inflammatory cells. Finally, 12% of joints were scored as normal with no signs of the initiation of synovitis, thus indicating the powerful suppressive effect of this therapeutic approach on inflammatory processes. FGK45 treatment completely protects 24% of the joints, and 29% were only mildly affected showing little erosion of the cartilage (Figure 4d). The remainder of the joints showed bone erosion and accumulation of inflammatory cells. The histological analysis of all the joints studied is reported in Figure 4e. Therapeutic administration of anti-CD40 mAb preferentially induces IgGl CII specific immunoglobulins.

Anti-CII antibodies are a trademark of CIA (collagen-induced arthritis), and have been reported to play a pathogenic role in the evolution of arthritis 19. In CIA, as in other models of autoimmune disease, a Thl self-antigen specific response drives the immune response . Therefore, upon CII in CFA immunisation high levels of CII specific IgG2a

antibodies, a Thl dependent subclass 21 , are produced. The isotype subclasses are an accepted indicator of whether a dominant Thl or Th2 response is mounted against CII in CIA. Thus the variation of the Thl (IgG2a) or Th2 (IgGl) responses is generally monitored in mice after therapeutic manipulations to evaluate possible in vivo shifts from a

22 23 pathogenic Thl (IgG2a) to a potentially protective Th2 (IgGl) response ' . Peripheral blood serum CII specific IgGl and IgG2a levels were quantified 12 days after disease onset (Figure 5). Both agonistic anti-CD40 mAbs upregulated the CII specific IgG2a (Thl) levels by 29 (FGK45) and 72 (3\23) fold, relative to control treated mice. Critically, the levels of IgGl (Th2) were amplified up to 46 fold for FGK45 and 260 fold in 3\23 treated mice. These data suggest that anti-CD40 mAb therapy would preferentially redirect the IgG2a/IgGl CII specific antibody ratio in favour of the Th2 controlled IgGl isotype.

Anti-CD40 mAb therapy alters the antigen specific T cell cytokine profile.

The increase of anti-CII IgGl/IgG2a ratio, observed after anti-CD40 mAbs treatment, suggests that anti-CD40 mAb therapy may have altered the T lymphocyte Thl/Th2 balance, therefore we studied the expression of Thl (IFN-γ) and Th2 (IL-4 and IL-5) cytokines in the different groups of mice. CII re-stimulation of splenocyte cultures (SPC) obtained from mice treated in vivo with 3\23 showed a significant inhibition of IFN-γ (Figure 6a), whereas the levels of IL-5 production were increased relative to the pattern observed in the isotype control treated mice (Figure 6c). In SPC from FGK45 treated mice a tendency to inhibit IFN-γ was observed, but no IL-5 production was detected (Figure 6a, c). Under these experimental conditions IL-4 was not detected in any of the samples studied (Figure 6b).

We also tested whether in vitro manipulation with the anti-CD40 mAb could influence the Thl/Th2 balance. In these experimental conditions in vitro re-stimulation of SPC from AFRC-MAC-1 treated mice (controls) with CII and 3\23, led to a significant reduction of IFN-γ production (Figure 6d) and to an increase of IL-5 and for the first time detection of IL-4 (Figure 6e and f). Of interest was the finding that the in vitro addition of 3\23 to SPC from 3\23 treated mice, further reduced the IFN-γ production (Figure 6d), paralleled by an increase in the IL-5 and more remarkably IL-4 levels (Figure 6e, f), compared to the value obtained from in vivo AFRC-MAC-1 treated SPC. n vitro challenge of SPC from AFRC-MAC-1 treated mice with FGK45 reduced the IFN-γ production (6d) and up-regulated IL-5 production (6f). These results indicate that agonistic anti-CD40 mAb therapy can effectively alter a pathogenic antigen- specific T cell phenotype and redirect, particularly in the case of 3\23, the auto- aggressive pro-inflammatory Thl response into an anti-inflammatory Th2 response.

Agonistic anti-CD40 administration before immunisation does not prevent CCIA.

It has recently been established that stimulation with agonistic anti-CD40 mAb can

13 1 R enhance T cell-independent . and T cell dependent immune responses . Since our data were in apparent disagreement with several well-established studies, we speculated that it was the timing of anti-CD40 mAb administration, which could be the crucial factor for the kind of immune response observed. Indeed the studies in which CD40 stimulation proved to amplify the immune response, the agonistic anti-CD mAb was introduced prior to ¹⁸ or

13 at the time of immunisation. To establish whether administration of anti-CD40 mAb before CII immunisation influenced the outcome of the disease we treated mice with 5 daily injections (300 μg/mouse) of FGK45, 3\23, AFRC-MAC-1 or PBS (300 μl/mouse) prior to CII in CFA immunisation. Thus we implemented an experimental protocol even more stringent to the one used by others prior immunisation ¹³' ¹⁸. In these experimental conditions both FGK45 and 3\23 did not prevent the induction of CCIA (Figure 7). Thus indicating, in our experimental model, that the timing of the administration of anti-CD40 mAbs appears to be crucial for the outcome of its effect.

Figure 8 shows that anti-CD40 antibodies reduce Ig G2a (THl pathogenic) levels in favour of Ig Gl (TH2) in splenocyte. Cells were treated with AFRC macl isotype monoclonal antibody or anti-CD40 monoclonal antibodies. The cells were then placed in SCID mice for one month before assaying for Ig G2a or Ig Gl .

Figure 9 shows the effect of injecting anti-CD40 antibodies into mice on IL-12 productions in pathogenic/arthrlitogenic splenocytes.

DISCUSSION

Disruption of the CD40-CD154 axis has recently gained central stage as one of the ideal approaches to control unwanted immune responses , more interestingly even other therapeutic strategies, such as the use of non depleting anti-CD4 mAb, seem to be based on down-modulation of CD 154 expression (unpublished results). Clearly the engagement of CD40 by agonistic mAbs provides the opposite effect of boosting the immune response, a property widely used to enhance the immune system when required ^" ' . However, in the course of earlier studies with non-depleting anti-CD4 mAb, the inventors paradoxically observed that agonistic anti-CD40 mAb could successfully prevent CCIA (unpublished results).

The results described here further support and expand this surprising finding establishing the therapeutic effect of agonistic anti-CD40 mAb in an autoimmune inflammatory animal model of RA. It is most remarkable that the agonistic anti-CD40 mAb therapy seems to be extremely effective in controlling the progression of ongoing CCIA. In our experimental model we could detect that a predominant CII specific Th2 response was induced in the anti-CD40 mAb treated mice suggesting that one of the mechanisms instigated by agonistic anti-CD40 mAb involves the induction of an autoantigen specific Th2 response.

One possible explanation for this distinct change in the profile of the T cell response, from a Thl to a Th2, is based on the potential shift of the cell type which acts as the dominant antigen presenting cell (APC) driving the expansion and activation of CII specific T cells. It is well established that presentation of antigen to T cells by DC tend to lead, in particular in the presence of 'danger' , to a Thl type of response . On the contrary, if non-resting

B cells act as APC they induce the activation of T cells expressing IL-4 with a Th2

compatible cytokine profile . The induction of these Th2 responses have been well

established to antagonise cell mediated immune responses ^" . Moreover, administration of Myelin Basic protein (MBP) peptide covalently linked to anti-IgD, to force presentation of the immunogenetic peptide by B cells, prevented the induction of a cell-mediated 32 autoimmune disease in rats . This prevention was subsequently shown to be due to a protective Th2 response and an alteration in the migratory properties of autoantigen

33 specific T lymphocytes . In this context it is essential to note that agonistic anti-CD40 mAb treated mice show a dramatic expansion of B cells and that a large proportion of these B cells are likely to be CII specific as large amounts of CII specific antibodies are present in these mice. Therefore the overwhelming APC population in the anti-CD40 mAb treated mice are likely to be the non-resting CII specific B cells. In addition it has to be reminded that antigen specific B cells are probably the most efficient APC, in terms of capturing ability ⁴, thus two factors, large expansion of non-resting B cells, and their antigen specificity would synergise and completely alter the balance of CII presenting APCs. It is also important to note in this respect that CD40 engagement stimulates B cell antigen

35 processing and hence enhance their APC function . Further supporting the hypothesis that agonistic anti-CD40 mAb manipulation, during an ongoing autoimmune chronic inflammatory process, can favourably redirect the type of immunological response to the driving autoantigen.

It is, however, apparent from our study that a simple switch from a Thl to a Th2 type of response could not account for the entire therapeutic action of anti-CD40 mAb. Firstly, the rapid effect after injection of agonistic anti-CD40 mAb seems to be unlikely only related to a switch of the dominant APC population. Secondly, the subtle but persistent differences between the effects of FGK45 and 3\23 indicate that alternative as yet undefined therapeutic mechanisms act in our model. In addition, the apparently least powerful Th2 inducer, FGK45, displays a potent therapeutic effect, possibly more so than that of 3\23. These discrepancies between the two antibodies, which were shown to recognise different epitopes on CD40 (data not shown), indicate that fine differences between these reagents create a distinct functional activity.

However, the paradox of the dual ability of agonistic anti-CD40 mAb to enhance immune responses, which proved to enforce a protective immune response to cancer ^14"16 and

13 infectious agents , as well as control ongoing inflammatory autoimmune processes, as reported here, remains to be reconciled. We suggest that the timing of administration ι o might be the key to explain such a difference, a view supported by others . The inventors have shown that the administration of anti-CD40 mAb before immunisation, compared to after antigenic challenge, does not produce the same functional outcome. This differential action of anti-CD40 mAb might be a good presage for the application of anti-CD40 mAbs to fight infections or cancer and alternatively, to treat chronic autoimmune inflammatory processes. Indeed in the first case the treatment will be properly timed prior to or at the time of antigenic challenge aiming to support the initiating response whilst in the second case we would interfere with an ongoing process aiming to deviate the established antigenic perception.

In conclusion, the inventors have described the totally unexpected finding that agonistic anti-CD40 mAbs can successfully control chronic autoimmune inflammatory processes opening their potential use for patients suffering from autoimmune disorders for whom to date no satisfactory therapy is available.

The results demonstrate that agonistic anti-CD40 antibodies are effective in controlling immune responses when there is already an immune response in place. For example, in immune-inflammatory responses in rheumatoid arthritis and other chronic autoimmune diseases.

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Claims

1. Use of an agonistic CD40 binding protein in the manufacture of a medicament to treat an autoimmune disease.

2. A method of treating an autoimmune disease comprising administering to an animal or patient a pharmaceutically effective dose of an agonistic CD40 binding protein.

3. A use or method according to claim 1 or 2, wherein the autoimmune disease is selected from rheumatoid arthritis, systemic lupus erythematosis, diabetes myellitus and multiple sclerosis.

4. A method or use, according to any preceding claim, wherein the agonistic CD40 binding protein is an anti-CD-40 antibody.

5. A method or use according to claim 4, wherein the anti-CD40 antibody is a polyclonal antibody or a monoclonal antibody.

6. A method or use according to claim 5, wherein the antibody is a monoclonal antibody with the functional characteristics of FGK45 or 3\23, when implemented in man, or CD 154.

7. A method of studying an autoimmune disease comprising administering a pharmaceutically effective dose of an agonistic CD40 binding protein to an animal, sample of tissue, or cell and observing a physiological change to the animal, sample of tissue or cell.

8. A method according to claim 7, wherein the CD40 binding protein is an anti-CD40 antibody.

9. A method according to claims 7 or 8, wherein the method is carried out in vitro on an isolated cell, cultured cell, or an isolated sample of tissue.