WO2001030974A2

WO2001030974A2 - Molecules targeting cd40 which do not activate cd40-expressing cells

Info

Publication number: WO2001030974A2
Application number: PCT/US2000/041532
Authority: WO
Inventors: Nancy Chang
Original assignee: Tanox, Inc.
Priority date: 1999-10-25
Filing date: 2000-10-25
Publication date: 2001-05-03
Also published as: WO2001030974A3; AU2471101A

Abstract

Disclosed are molecules which are not antibodies which bind to the CD40 antigen but which do not cause proliferation, differentiation or activation of B lymphocytes or other cells carrying the CD40 antigen and can inhibit CD40 ligand induced activation. These molecules target, bind to or interact with a particular epitope on CD40. They are useful for treating autoimmune, cell-mediated and antibody-mediated diseases.

Description

Molecules Targeting CD40 Which Do Not Activate CD40-Expressing Ceils

Field of the invention The invention relates to non-antibody molecules which target, bind to or interact with an epitope on CD40 and which do not activate CD40-expressing cells.

Background of the Invention

The CD40 antigen is a 50 kDA type 1 membrane glycoprotein expressed on the B cell surface, as well as on other cells. It is critical for B cell functions, including proliferation, differentiation, rescue from apoptosis and isotype switching, which are induced when CD40 binds to CD40 ligand ("CD40L").

CD40L is primarily located on activated T cells. Interference with CD40 activity is potentially beneficial for antibody-mediated diseases such as autoimmunity, allergic diseases and conditions in which imunogenic proteins are used therapeutically, such as in treatment with exogenous blood products or in gene therapy.

CD40 is expressed by a variety of cell types, including antigen presenting cells, for example, macrophages and dendritic cells, where it functions in activating these cells. Interference with CD40 activity could therefore be beneficial in treatment of cell-mediated immunological diseases, including multiple sclerosis ("MS"), psoriasis, idiopathic thrombocytopenia purpura ("IBD") and transplantation. The in vivo role of the CD40/CD40L interaction has been demonstrated in

animal models using anti-CD40L treatment, CD40 or CD40L knock-out animals,

or animals transgenic for CD40L expression. As expected, interference with this

interaction reduces signs and symptoms of collagen arthritis, lupus, nephritis, graft-versus-host disease, experimental allergic encephalomyelitis ("EAE") and

allergic contact dermatitis, as well as increasing the survival of allografts.

Cross-linking of CD40 molecules with anti-CD40 antibodies usually

results in B cell activation. However, antibodies have been developed which

bind to CD40 and which do not stimulate the growth and differentiation of B

cells, and can inhibit B cell responses. See U.S. Patent Nos. 5,677,165 and

5,874,082. These antibodies are potentially useful in treatment of autoimmune,

cell-mediated and antibody-mediated diseases. Summary of the Invention

The invention includes non-antibody molecules which target, bind to or

interact with an epitope on CD40 and which do not do not stimulate the growth, activation and differentiation of one or more of B cells, monocytes and dendritic

cells, and can inhibit CD40 ligand induced activation. The molecules can inhibit

B cell responses. They may have the additional property of not interfering with

binding of CD40L to the epitope. One example of such an epitope on CD40 is that bound by the antibody

designated 5D12. This epitope is at amino acid residue numbers 52-63 of the

CD40 antigen sequence (See SEQ ID NO:1 ). A model of the CD40 antigen shows that this epitope is on the opposite side of CD40 from where the CD40 ligand binds. Amino acids implicated in the binding of CD40L binding are located in the region of amino acid residue numbers 70 to 120 of CD40. See Fig. 1. The non-antibody molecules of the invention include peptides, oligonucleotides, and other chemical entities which are not antibodies. They are useful for in interrupting the CD40/CD40L interaction and in treatment of autoimmune, cell-mediated and antibody-mediated diseases. Brief Description of the Drawings Fig. 1 shows, in schematic form, the putative binding site of the monoclonal antibody 5D12, and the CD40L binding site on CD40.

Fig. 2 is a FACS graph showing that a saturating amount of SD12 does not affect binding of CD40L-FITC.

Fig. 3 is a FACS graph showing that pre-incubation of B cells with other anti-CD40 antibodies can prevent binding of CD40L-FITC. Brief Description of the Sequence Listing

SEQ ID NO:1 represents the sequence of the CD40 antigen.

SEQ ID NO:2 represents the sequence of the epitope where the anti- CD40 molecules of the invention bind. Detailed Description of Making and Using the Invention

The molecules described and claimed include peptides and other chemical entities which are not antibodies. They can be isolated or screened from compound libraries by conventional means. An automated system for

generating and screening a compound library is described in U.S. Patent Nos.

5,901 ,069 and 5,463,564. A more focused approach involves three-

dimensional modeling of the binding site, and then making a family of molecules

which fit the model. These are then screened for those with optimal binding characteristics.

Another approach is to generate recombinant peptide libraries, and then

screen them for those which bind to the epitope of CD40 of interest. See, e.g.,

U.S. Patent No. 5,723,322. Molecules can, in fact, be generated or isolated

with relative ease in accordance with techniques well known in the art based on the epitope being known. The molecules can be administered by any of a number of routes. In the case of peptides, because they are subject to

degradation in the gastro-intestinal tract, they would preferably be injected.

Other compounds of the invention could also be injected. The injections could

be intra-muscular, intra-venous or sub-cutaneous.

Non-peptide molecules of the invention could be administered orally,

including by suspension, tablets and the like. Liquid formulations could be

administered by inhalation of lyophilized or aeorosolized microcapsules.

Suppositories could also be used. Additional pharmaceutical vehicles could be used to control the duration of action of the molecules of the invention. They could be entrapped in

microcapsules prepared by coacervation techniques or by interfacial polymerization (hydroxymethylcellullose or gelatin microcapsules) in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.

Excipients, for example, salts, various bulking agents, additional buffering agents, chelating agents, antioxidants, cosolvents and the like can be included in the final formulation. Specific examples include tris-(hydroxymethyl) aminomethane salts ("Tris buffer") and disodium edetate.

The dosage and scheduling for the formulation which is selected can be determined by standard procedures, well known in the art. Such procedures involve extrapolating an estimated dosing schedule from animal models, and then determining the optimal dosage in a human clinical dose ranging study.

Such a dose ranging study could also monitor a variety of indicators related to the CD40-CD40L pathway, including a decrease in B lymphocytes, monocytes or dendritic cells, a decrease in free immunoglobulin and effect on disease symptoms. Adverse effects and side-effects would also be monitored.

The in vivo effect of the molecules of the invention can be extrapolated from the known effects of anti-CD40 antibodies which do not cause proliferation or differentiation of cells carrying CD40. One of these anti-CD40 antibodies, designated 5D12, has been extensively studied, as described below. 5D12 binds to the epitope which is targeted, bound or interacted with by the molecules of the invention. 1. Location of the 5D12 Binding Epitope and the CD40L Binding Epitope

The reactivity of 5D12 with a panel of overlapping synthetic peptides corresponding to the amino acids sequence of the extracellular domain of CD40 was tested. Because Mab 5D12 binds poorly to CD40 when tested in Western blotting, some control experiments were performed to see if 5D12 would still bind to denatured CD40 in an ELISA system. CD40-lg was coated onto ELISA plates by drying at 37°C overnight or by incubation overnight in PBS at 4°C. In each case, CD40-lg was pre-treated by boiling for 10 minutes and/or with 1 mM DTT.

These pilot experiments demonstrated that boiling the antigen before coating did not significantly decrease the binding of Mab 5D12. However, reduction of all disulphide bonds in CD40-lg strongly reduced the binding of Mab 5D12. Since a weak signal remained under these conditions, it was decided to proceed with the Pepscan analysis, which showed that Mab 5D12 strongly reacted with one specific 12-mer peptide of the extracellular part of CD40. See SEQ ID NO:2 This peptide corresponds to amino acids 32 to 43 of the mature protein. At this position in the CD40 sequence there is a high degree (90%) of homology with CD40 from non-human primate species. In contrast, the degree of homology with mouse and bovine CD40, to which 5D12 does not bind, is only 42% and 58%, respectively. Interestingly, this peptide is distantly located from the CD40L binding site on CD40. Amino acids in CD40 that have been implicated in binding to CD40L are located in the region of amino acids 70 to 120 of the mature protein. It appears that the CD40L-CD40 interaction is concentrated on at least two clusters of residues on CD40 and it is predicted that CD40L-CD40 contacts are formed along the interface of two CD40L monomers with one CD40 chain (Bajorath et al., Biochemistry 34:9884 (1995)). Fig. 1 shows the location of the putative 5D12 binding epitope on a model of the extracellular domain of CD40 (Bajorath and Aruffo, Proteins 27:59 ((1997)). In this model the amino acids 32-43 are highlighted in bold and a number of residues postulated to be involved with CD40L binding indicated by dotted lines. This model clearly demonstrated that the putative CD40 binding epitope is located on the "outside" of the CD40 molecule, the "outside" being based on the hypothesis that three CD40 monomers bind around one CD40L trimer.

2. CD40L Binds to Another Location on CD40 from 5D12; 5D12 Seems to Affect CD40L Signaling

Preliminary two-color FACS analysis showed that 5D12 and an FITC- labeled soluble trimeric CD40L (CD40L-FITC) could simultaneously bind to CD40-expressing cells. The hypothesis that 5D12 binds to a distinct epitope from where CD40L binds was also tested in additional experiments. Pre- incubation of JY B cells with or without CD40L-FITC did not affect the staining intensity obtained with a saturating amount of 5D12. The reciprocal experiment showed that a saturating amount of 5D12 did not affect subsequent binding of CD40L-FITC (Fig. 2). In contrast, pre-incubation of JY B cells with other anti- CD40 monoclonal antibodies (one of which is designated G28.5) could prevent subsequent binding of CD40L-FITC (Fig. 3).

The disappearance of 5D12 and CD40L from stained JY B cells was investigated over time. When JY B cells labeled with CD40L-FITC were washed

and subsequently cultured at 37°C, the fluorescent signal decreased over a

period of hours. The release of CD40L-FITC from the cell surface was at about the same rate when the CD40L-FITC loaded cells were cultured in the presence of 5D12. Furthermore, in a reciprocal experiment, the level of CD40 on JY B cells did not appear to be significantly altered during culture with 5D12, nor did pre-binding CD40L-FITC to the cells affect the level of CD40 detected using 5D12.

In summary, these experiments clearly show that 5D12 in vitro: (i) does not compete with CD40L for binding to CD40; (ii) does not cause the release of CD40L bound to CD40; and (iii) does not cause modulation of CD40 from the cell surface. Previous results showed that the inhibitory effect of 5D12 on CD40 dominates over the stimulatory effect of CD40L. 5D12 may be modulating CD40 in such a way that signaling via CD40 is prevented or aborted when CD40L has already engaged CD40. 3. 5D12 Inhibits CD40L Mediated Activation

In a THP-1 assay, the effects of murine 5D12 on IL-8 production, induced

by a number of different stimuli that are known to signal via NFKB, were tested. It was found that at concentrations where Mab 5D12 completely inhibit CD40L- mediated IL-8 production, there was no effect on IL-8 production by THP-1 cells

stimulated with LPS, TNFα, PMA or ionomycin, which normally induce IL-8

production.

4. Anti-CD40 Shows Efficacy in an EAE Model of Multiple Sclerosis

An animal model of experimental autoimmune encephalomyelitis ("EAE") showed that treatment with the chimeric version of 5D12 (ch5D12) after disease

induction significantly delayed the induction and reduced the severity of the EAE. EAE was induced in marmoset monkeys with myelin oligodendrocyte

glycoprotein (MOG), a minor component of the outer layer of the myelin sheath.

Anti-MOG antibodies have been implicated in MS and can be found in active

inflammatory lesions of MS patients (Genain et al., Nature Medicine, 5:170 (1999)).

Marmoset monkeys were immunised with 100 μg recombinant MOG as

an emulsion in CFA. Under ketamine sedation, the animals were injected

intradermally in the back with 600 μl of emulsion divided over 4 locations. The

monkeys were divided in two groups of 4 monkeys, with each receiving 5 mg/kg

of body weight ch5D12 or placebo 2 to 3 times per week, starting one day

before disease induction, for a period of 50 days. The serum half-life of ch5D12 was unexpectedly low in the marmoset

monkeys. It was observed in 3 out of the 4 animals that when the dosing

frequency of ch5D12 was reduced from 3 times per week to 2 times per week (after day 10), not all circulating CD40⁺ cells remained saturated with ch5D12. It was therefore decided at day 28 to increase the dosing frequency back to 3 times per week. The circulating free ch5D12 serum concentrations ranged from

10 to 40 μg/ml during the first 2 weeks, and then dropped below 10 μg/ml in 3

out of 4 animals after decreasing of the dosing frequency. Only the animal that did not experience a drop in its free circulating ch5D12 concentrations below 10

μg/ml showed a continuous saturation of expressed CD40 with ch5D12.

Increasing the dosing frequency resulted only in a slight increase in circulating

ch5D12 concentrations in the 3 animals which had dropped below 10 μg/ml.

Only the animal which did not drop below the 10 μg/ml and had continued

saturation of expressed CD40 (QN) showed increased serum concentrations of ch5D12 when the dosing frequency was increased back to 3 times per week.

From a small toxicology experiment preceding this study, it was known that ch5D12 gave rise to an anti-ch5D12 antibody response about 12 to 17 days after a single administration of 5 mg/kg. When the ch5D12 treated marmoset monkeys were tested for anti-ch5D12 antibodies, it was found that the 3 animals

in which circulating ch5D12 concentrations had dropped below 10 μg/ml

developed an anti-ch5D12 response starting at days 17 to 27, depending on the animal. There was a correlation between the circulating ch5D12 concentration, saturation of all CD40 with ch5D12 and the development of an anti-ch5D12 response. The animals developed an anti-ch5D12 response in the same order

in which the circulating ch5D12 concentrations dropped below 10 μg/ml. In an animal which did not develop an anti-ch5D12 response during the first 35 days

of the study, the circulating ch5D12 concentrations did not drop below 10 μg/ml. Only one animal showed a complete saturation of all CD40 in vivo for the whole 50 day period. All four animals in the placebo group had developed EAE by day 4, with the first animal developing EAE at day 32. In contrast, none of the ch5D12 animals had clinical signs of EAE at day 41. In the placebo group, 2 animals were terminated before day 50 because of the severity of their disease. On day 50, according to the protocol, the remaining 2 animals of the placebo-control group and 2 out of 4 of the ch5D12-treated animals were sacrificed for histological analysis. The two other ch5D12-treated animals were kept alive. One of these ch5D12-treated animals developed EAE approximately one week after cessation of the treatment. The other ch5D12-treated animal was still free of clinical signs at day 100. It was shown that treatment with ch5D12 had a strong inhibitory effect on the induction of anti-MOG antibodies. Preliminary MRI analysis of post mortem brains revealed that all placebo-treated animals had a very high lesion load when they were sacrificed (due to disease severity or at day 50, according to the protocol). The ch5D12-treated animal that developed EAE shortly after ch5D12 treatment was stopped also had a high lesion load. In contrast, the two ch5D12-treated animals that were sacrificed at day 50 only had minor inflammatory lesions, as visualised by the post-mortem MRI. This demonstrates

Claims

the involvement of the CD40L-CD40 interaction in the formation of inflammatory lesions in this disease model. Furthermore, this experiment demonstrated that blocking the CD40L-CD40 interaction with saturating concentrations of ch5D12 effectively prevents induction of EAE in marmoset monkeys. This biological effect seems to correlate with the effects on the induction of anti-MOG antibodies.It should be understood that the terms and expressions used herein are exemplary only and not limiting, and that the scope of the invention is defined only in the claims which follow, and includes all equivalents of the subject matter of those claims.What is Claimed Is:

1. Molecules which are not antibodies which bind to CD40 and which do not cause proliferation, differentiation or activation of CD40-expressing cells but inhibit CD40L induced activation.

2. Molecules of claim 1 which target, bind to or interact with the epitope represented by SEQ ID NO:2.

3. Molecules which are not antibodies which bind to CD40 but which do not interfere with the binding of CD40L to CD40.

4. A pharmaceutical composition comprising molecules which are not

antibodies which bind to CD40 but which do not cause proliferation,

differentiation or activation of CD40-expressing cells.

5. The pharmaceutical composition of claim 4 wherein the molecules target, bind to or interact with the epitope represented by SEQ ID NO:2.

6. A pharmaceutical composition comprising molecules which are not antibodies which bind to CD40 but wherein the molecules do not interfere with the binding of CD40L to CD40.

7. A method of treating autoimmune, cell-mediated and antibody-mediated diseases, and diseases and conditions in which imunogenic proteins are used therapeutically, comprising administering the molecules of claims 1 or 2.

8. A method of treating autoimmune, cell-mediated and antibody-mediated diseases, and conditions in which imunogenic proteins are used therapeutically, comprising administering the molecules of claim 3.