WO1999038531A1

WO1999038531A1 - TREATING ATOPIC DERMATITIS WITH IgE ANTAGONISTS

Info

Publication number: WO1999038531A1
Application number: PCT/US1999/000135
Authority: WO
Inventors: Tse Wen Chang
Original assignee: Tanox Inc
Current assignee: Tanox Inc
Priority date: 1998-01-29
Filing date: 1999-01-06
Publication date: 1999-08-05
Anticipated expiration: 2000-07-29
Also published as: EP1056471A1; JP2002501905A; AU2103699A; CN1289253A; EP1056471A4

Abstract

The invention relates to a pharmaceutical composition for treatment of atopic dermatitis comprising a suitable IgE antagonist which does not induce release of the mediators of allergy; for example, anti-IgE antibodies which bind to secreted IgE, to membrane IgE on the surface of IgE-producing B cells, but not to IgE bound to the FcεRI on the surface of basophils or mast cells. Preferably, these antibodies also do not bind to IgE bound to FcεRII receptors. It is also preferable if these antibodies have human IgG1 or IgG3 constant regions, as well as further human portions, if desired. The pharmaceutical composition can be administered systemically or topically.

Description

1 Treating Atopic Dermatitis with IgE Antagonists

Field of the Invention

The invention relates to use anti-IgE antagonists, including monoclonal

antibodies, and their use in treating atopic dermatitis.

Background of the Invention

Immunoglobulin E (IgE) is one class of immunoglobulin (or "antibody")

molecule. IgE is present in human serum in lower concentrations than the other

immunoglobulins: IgG, IgM, IgA, and IgD. IgE is thought to have a role in protection

against parasites, but has never been definitively established as playing a

necessary, or even a beneficial role, at least in developed countries, where parasite

infections are not a significant problem. IgE is well known as the mediator of

immediate-type hypersensitivity allergic reactions, including allergic rhinitis ("hay

fever"), extrinsic asthma, and food and drug allergies.

In IgE-mediated allergic reactions, IgE, after it is secreted by B cells, binds

through its Fc portion to the FcεRI receptors, which are present on the surface of

basophils, mast cells and Langerhans cells. If the IgE bound to the surface of these

cells now contacts and binds an allergen, this causes a cross-linking of the bound

IgE molecules and hence the underlying receptors, and triggers the release of

pharmacologic mediators, such as histamine, serotonin, leukotrienes and the slow-

reacting substance of anaphylaxis. These mediators cause the pathologic

manifestations of allergic reactions.

Some patients with a history of some or all of the IgE-mediated allergic 2 conditions also suffer from a painful skin condition called atopic dermatitis. Atopic

dermatitis is characterized by pruritis (itching), redness, and painful skin lesions.

In some patients with chronic dermatitis, the skin can become lichenified. While

atopic dermatitis is often found together with other allergic diseases, no correlation

has been established between high IgE levels and the severity, or the

manifestation, of atopic dermatitis in affected patients. Those in the field believe

that atopic dermatitis may have a link to IgE, but that IgE alone is not the causative

agent.

A particular class of anti-IgE antibodies has been developed to treat allergic

diseases. These antibodies bind to secreted IgE, but not to IgE attached to the

FcεRI receptors. When these anti-IgE antibodies are administered internally, they

bind to IgE and neutralize it. Preventing its binding to either FcεRI or FcεRII

receptors, the latter being present on B cells and other cell types as well. These

anti-IgE antibodies also bind to IgE which is attached to the membrane of IgE-

producing B cells (the "membrane form of IgE"). By doing so, they may further aid

in down-regulating or eliminating, through antibody dependent cellular cytotoxicity

("ADCC") or complement mediated cytolysis, the IgE-producing B cells, and

therefore, reduce the levels of secreted IgE. Because they do not bind to IgE

attached to the FcεRI, however, they do not cause cross-linking and do not

themselves result in release of pharmacologic mediators of allergy.

It has been shown that such anti-IgE antibodies can lower IgE levels, in both

animal models and human clinical trials. See Come et al., J. Clin. Invest. 99, No. 3 5, 879-887 (1997). Such anti-IgE antibodies also demonstrated efficacy in treating

allergic rhinitis and extrinsic asthma in several human clinical trials, as would be

expected from the fact that they lower IgE levels. See Come et al., id.; Boulet et al.,

Am J. Respir. Crit. Care Med., 155: 1835-1840 (1997); Fahy et al., J. Respir. Crit.

Care Med., 155: 1828-1834 (1997). No clinical trials of these anti-IgE antibodies

have been performed for treatment of atopic dermatitis. Based on the lack of

correlation between high IgE levels and severity of atopic dermatitis symptoms, it

would not be expected that these antibodies would be useful in atopic dermatitis

treatment. Regarding other IgE antagonists which function by preventing or

inhibiting IgE from binding to the FcεRI without inducing release of the

pharmacologic mediators of allergy, they would also not be expected to be an

effective atopic dermatitis treatment. Such antagonists would include small

molecules and other new chemical or biological entities.

Summary of the Invention

The invention includes a pharmaceutical composition for treating atopic

dermatitis comprising IgE antagonists which do not induce release of the

pharmacologic mediators of allergy. Such antagonists include monoclonal anti-IgE

antibodies which bind to secreted IgE but not to IgE bound to the FcεRI receptors

present on basophils, mast cells, or Langerhans cells. The anti-IgE antibodies

preferably also bind to membrane IgE, and thereby aid in down-regulating or

eliminating IgE-producing B cells, leading to further reduction in secreted IgE levels.

These anti-IgE antibodies preferably do not bind to IgE bound to the low affinity 4 FcεRI I receptors. If the antibodies of the invention did bind to IgE bound to the

FcεRI I receptors, they could cause the destruction or down-regulation of B cells

producing other classes of immunoglobulins, or other cell types, which would be

undesirable.

Notwithstanding the conventional wisdom that depleting or removing IgE,

without doing more, will not be effective in treatment of atopic dermatitis, this patent

application rests on the premise that IgE antagonists including anti-IgE alone, will

prove an effective treatment. In fact, anti-IgE should prove to be a substantial

benefit to patients with atopic dermatitis, well beyond the expectations of those in

the field.

If the IgE antagonist chosen is anti-IgE antibody, it can be modified in order

to make it less antigenic and more suitable for human administration by techniques

including chimerization, humanization (through CDR-grafting), or otherwise,

including producing fully human antibodies produced in transgenic animals or single

chain fragments of human antibodies produced through phage display library

technology.- Preferably, the antibodies have a human lgG1 or lgG3 constant heavy

chain region, as such regions are known to mediate ADCC and complement

mediated cytolysis, thereby aiding in elimination of IgE-producing B cells. The

pharmaceutical composition of the invention is likely to be most effective when

internally administered, such as by intravenous, intramuscular, or subcutaneous

injection. It could also be internally administered by oral ingestion, in a suitable

carrier which is not subject to digestive degradation, or through the alveoli of the 5 lung by an inhaler. It may also be possible to administer the composition topically

90 to affected areas, where it would be absorbed and act locally.

Description of Making and Using the Invention

1. Making the Various Embodiments of the Invention

Chemical or biological entities suitable for use as IgE antagonists can be

selected and screened by a number of methods, including using assays similar to

95 those used to screen TES-C21 , described below. In essence, one would screen

first for those that bound to secreted IgE, and then, from that group, those that did

not induce release of pharmacologic mediators of allergy would be selected. A

number of different assays, well known to those in the art, could be used to

accomplish this.

100 In one specific embodiment, the monoclonal anti-IgE antibodies used with

this invention are produced by continuous (immortalized), stable, antibody-

producing cell lines. The preferred antibody-producing cell lines are hybridoma and

transfectorηa cell lines. However, they can be any cell lines which contain and are

capable of expressing functionally rearranged genes which encode the antibodies

105 (or fragments) of interest. Lymphoid cells which naturally produce assembled

immunoglobulin are preferred.

Hybridoma cells which produce the specific antibodies used with this

invention can be made by the standard somatic cell hybridization technique of

Kδhler and Milstein, Nature 256:495 (1975) or similar procedures employing

110 different fusing agents. Briefly, the procedure is as follows. The monoclonal anti- 6

IgE antibodies are produced by immunizing an animal with human IgE or IgE-

producing B cells, or peptidic segments of human IgE (secretory or membrane),

which are identified as including the epitope of interest, which is in the Fc region of

IgE. Peptides can be synthesized or produced by recombinant DNA technology

115 and, for enhanced antigenic effect, conjugated to a carrier protein, such as keyhole

limpet hemocyanin. Following immunization, lymphoid cells {e.g., splenic

lymphocytes) are obtained from the immunized animal and fused with immortalizing

cells {e.g., myeloma or heteromyeloma) to produce hybrid cells. The hybrid cells

are screened to identify those which produce the desired anti-IgE antibody by

120 following the screening methods described below in detail.

It is preferred that, when long-term administration of antibodies is

contemplated, as it is here where anti-IgE is used for treating atopic dermatitis, the

antibodies be either human or substantially human, to reduce or eliminate the

human anti-mouse (HAMA) response. The murine antibody portions could

125 themselves trigger an allergic response, or the HAMA response against such

portions could reduce the effectiveness of the treatment.

Human hybridomas which secrete human antibodies can be produced by the

Kόhler and Milstein technique. Although human antibodies are especially preferred

for treatment of humans, in general, the generation of stable human-human

130 hybridomas by such techniques for long-term production of human monoclonal

antibody can be difficult. An alternative technique for producing human antibodies

is production in transgenic mice. Briefly, this approach involves disruption of 7 endogenous murine heavy and kappa light chain loci, followed by construction of

heavy and K light chain transgenes containing V, D, J segments, and C genes of

135 human origin. These are then introduced by pronuclear microinjection using human

transgenes. The mice are then cross-bred to generate the human antibody

producing strains. This technique is describe in more detail in, among other

references, U.S. Patent No. 5,569,825. The technology may be available under

license from GenPharm International, Inc. (Mountain View, California).

140 Another alternative for solving antigenicity problems is to produce fully

human antibody fragments, for example, the single chain Fv region, by the phage

display library methodology. Briefly, this involves amplification of the human V gene

repertoire from bone marrow, blood and tonsil samples by polymerase chain

reaction ("PCR"), followed by preparation of separate libraries containing heavy and

145 light chain (both K and λ) chain V genes. These separate fragments are then

assembled into a single chain Fv for display on the surface of phage, where the

desired fragments can be readily screened. References describing this technique

in more detail include U.S. Patent No. 5,565,332 and European Patent No. 0 589

877 B1. The technology may also be available under license from Cambridge

150 Antibody Technology Limited, Melbourn, England.

Production of antibodies in rodents, especially mice, is a very well

established procedure. An alternative to reduce the murine portions of the anti-IgE

antibodies is to produce them in a rodent system, and convert them into chimeric

rodent/human antibodies or CDR-grafted humanized antibodies by established 8

155 techniques. Chimeric antibodies can be produced as described, for example, in

U.S. patent No. 4,816,397. The making of humanized antibodies is described,

among other references, in U.S. Patent Nos. 5,693,762; 5,693,761 ; 5,225,539, and

in WO 89/06692 and WO 92/22653.

One example of an anti-IgE antibody of the invention (designated TES-C21 )

160 and its chimeric mouse-human form (designated TESC-2) is described in

International Application WO92/17207. The screening protocols (described below)

for TES-C21 and TESC-2 can be applied to other anti-IgE antibodies to yield

antibodies of the invention suitable for chimerization or humanization through CDR-

grafting. The hybridoma cell lines producing TES-C21 are available from the

165 American Type Culture Collection ("ATCC"), Rockville, Maryland under Accession

No. 11134, .and those producing TESC-2 are on deposit under Accession No. BRL

9

A humanized version of the murine antibody TES-C21 was made, as

described in detail in Australian Patent No. 675449, granted May 25, 1997. Similar

170 procedures can be followed to produce other humanized anti-IgE antibodies.

Several transfectomas producing humanized anti-IgE antibodies suitable for use

with the invention are available from the ATCC under the following accession

numbers: 11130; 11131 ; 11132; 11133. An anti-IgE antibody similar to that

produced from the transfectoma deposited under accession number 11131 is

175 among those with potential for full clinical development for treatment of atopic

dermatitis. Another humanized antibody suitable for treatment of atopic dermatitis

is E25 (rhuMAb-E25), produced by Genentech, Inc. This antibody is described in

Presta et al., J. Immunol. 151:2623-2632 (1993).

A. Production and Screening of TES-C21 and TESC-2

180 TES-C21 and TESC-2 were produced and screened as follows. Briefly, male

Balb/c mice were immunized several times with polyclonal human IgE from sera

(provided by Ventrex). The IgE was combined with a suitable adjuvant. Mice were

sacrificed after the last injection of immunogen and the spleens were removed for

preparing single cell suspensions for fusion with myeloma cells. The spleen cells

185 were fused with Sp2/0 cells using a fusion mixture of polyethylene glycol 1450

(Kodak), CMF-PBS and DMSO. DMEM was added after the cell suspensions were

combined.

The hybridomas resulting from the fusion were then screened by enzyme-

linked immunosorbent assay (ELISA) against human IgE bound to an Immulon 2 10

190 plate. One of these hybridomas produced TES-C21.

TES-C21 was further screened, by ELISA, to be specific for human IgE, and

not to react with IgG, IgM, IgA, IgD, human serum albumin, transferrin or insulin.

TES-C21 bound equally well to various human IgE molecules. TES-C21 bound to

the IgE-secreting cell lines SKO-007, U266 and SE44 in a dose-dependent manner,

195 indicating binding to human membrane IgE. But TES-C21 did not bind to human

B cell lines bearing surface IgM, IgD, IgG, or IgA, or to a T cell line, or to the parent

murine cell line of SE44, or to a murine cell line secreting chimeric human IgG.

TES-C21 also does not bind to IgE on high affinity FcεRI receptors or on low affinity

FcεRI I receptors which are present on a wide variety of cell types. It also did not

200 induce histamine release from freshly prepared human blood basophils, on which

the FcεR are armed with IgE. At 10 μg/ml TES-C21 is able to inhibit completely the

binding of 1 μg of IgE to FcεRII.

To generate TESC-2, Sp 2/0 cells were co-transfected with the variable

regions of TES-C21 H and L-chains, and human γ1 and K constant regions, and

205 aliquoted into 96 well plates for selection. Supernatants were screened for

secretion of human IgG which bound to human IgE. The transfectoma cells were

adapted to growth in serum-free medium. TESC-2 was then purified from medium

of confluent cultures using an immobilized protein A column.

TESC-2 and TES-C21 bind equally well to IgE bound to microtiter plates.

210 This was demonstrated as follows. Immulon 2 plates were coated with gp120 11 peptide-ovalbumin conjugate and lgE-SE44 was bound to the immobilized antigen.

TES-C21 or TESC-2 at various concentrations were added. Binding was detected

using either horseradish peroxidase ("HRP"), -conjugated goat antimouse IgG (for

TES-C21 ) or HRP-goat antihuman IgG, Fc (for TESC-2).

It was determined that TESC-2 and TES-C21 also have the same relative

affinity for IgE bound to microtiter plates. Immulon 2 plates were coated with gp120

peptide-ovalbumin conjugate and lgE-SE44 was bound to the immobilized antigen.

TES-C21 and TESC-2 at various concentrations were added and preincubated for

1 hour before adding 0.22 μg/ml of biotinylated TES-C21. Binding of biotinylated

TES-C21 was detected using horseradish peroxidase-conjugated streptavidin.

TESC-2 and TES-C21 also were shown to bind equally to IgE-producing

cells. This was demonstrated by incubating such cells at 2 x 10⁶ cells/100 μl PBS-

1 % goat serum at various antibody concentrations at 0° for 30 minutes. Binding of

TES-C21 was detected using FITC-goat (Fab')₂ antimouse IgG; binding of TESC-2

was detected using FITC-goat (Fab')₂ antihuman IgG. Binding was quantitated by

fluorescence flow cytometry using a Coulter Epics V. The FITC intensity gate was

set to yield 10% ± 0.5% positive cells in the absence of primary immunoglobulins.

It was found that neither TES-C21 nor TESC-2 bound to IgE which was

bound to low affinity IgE receptors. The possibility that TESC-2 recognized IgE

complexed with CD23 was studied using cells of an IgG-secreting human

lymphoblastoid line, IM-9. The presence of CD23 on IM-9 cells was confirmed by 99/38531

12 their strong staining with anti-Leu 20, a MAb specific for CD23. IM-9 cells were

incubated with 5 to 10 μg/ml of human IgE, washed, and then incubated with biotin-

labeled TESC-2 or a positive control anti-IgE MAb TES-19, followed by FITC-

streptavidin and analyzed by flow cytometry.

Both chimeric TESC-2 and murine TES-C21 were shown to inhibit binding

of IgE to FcεRII. The antibodies were preincubated at various concentrations with

20 μg lgE-SE44 for 1 hour at 37° before addition of IM-9 cells bearing FcεRII.

Binding of - IgE to cells was detected using biotinylated TES-19 and FITC-

streptavidin and quantitated by fluorescence flow cytometry.

To negate the possibility that immune complexes of TESC-2 and IgE, formed

during their preincubation in these experiments, were binding to cells but yielding

false negatives, it was confirmed that these immune complexes also did not bind

to FcεRII, using biotin-labeled TESC-2 or FITC goat anti-human IgE (with TES-

C21).

Neither TESC-2 nor TES-C21 induces histamine release from freshly

prepared human blood basophils on which the FcεR are armed with IgE. Due to the

variable release of mediators from basophils of different donors, the antibodies

were examined at multiple concentrations on basophil preparations from more than

50 individual donors. No induction of histamine release by TESC-2 or TES-C21

was observed.

To address the possibility that TES-C21 might bind to basophils but not 13 induce cross-linking of the receptors to induce histamine release, a secondary

antibody was used for crosslinking. Since anti-human IgG alone can induce

255 histamine release, only the murine antibody TES-C21 was used in these

experiments. The crosslinking goat antimouse IgG enhances histamine release

induced by suboptimal concentrations of control anti-lgEs. However, TES-C21 did

not induce histamine release even under these very permissive conditions.

TESC-2 was further tested to determine whether it could block the binding

260 of IgE to FcεRI receptors, and whether immune complexes of IgE and TESC-2

would bind to these receptors. To determine whether TESC-2 inhibits the binding

of human IgE to FcεRI, human peripheral blood basophils that had been depleted

of IgE by treatment at low pH were reloaded or sensitized with SE44-derived

chimeric IgE reactive to a peptide antigen. Functional binding of SE44 IgE was

265 tested by histamine release induced by the polyvalent R15K peptide-ovalbumin

conjugate to which the variable region of lgE-SE44 binds. Preincubation of IgE-

SE44 with TESC-2 inhibited IgE binding to FcεRI. Binding of SE44 IgE was also

inhibited when basophils were incubated with another IgE (PS) before exposure to

lgE-SE44. It may be assumed that immune complexes of TESC-2 and IgE were

270 formed during the preincubation and these also did not cause the release of

histamine. The experimental conditions and the results of these experiments are

summarized below in Table 1. 14

Table 1

Inhibition of IgE Binding to High-Affinity

275 IgE Receptors by TESC-2

Net Histamine Release (% of total)

Conditions for Basophil Challenge with R15K Loading with lgE-SE44 Peptide-Ovalbumin Challenge with Anti-IgE

lgE-SE44 was not preincubated 37 66 with TESC-2

lgE-SE44 was preincubated with 3 68 TESC-2

lgE-SE44 was preincubated with 0 63 IgE-PS

These studies have also been performed, and similar results obtained, with

the CDR-grafted version of TES-C21 referenced above.

280 2. Using the Antibodies of the Invention for Treatment of Atopic Dermatitis

Prior to commercial availability, the IgE antagonists, or antibodies, of the

invention must be subjected to human clinical trials to confirm their safety and

efficacy. A sample protocol for such a clinical trial would be to start with up to 30

patients affected by atopic dermatitis who continue to have symptoms despite

285 regular medical care for atopic dermatitis, including such treatments as anti-

histamines, corticosteroids, soothing baths and lotions. Patients would receive 15 intravenous or subcutaneous injections of 50 to 300 mg of anti-IgE at weekly, bi¬

weekly or monthly intervals for 3 to 6 months. Effects of anti-IgE treatment on their

ongoing atopic dermatitis would be scored, e.g., using the SCORAD index (see B.

290 Kung et al., Dermatology 1997, 195:10-19). The relationship between effect on

circulating IgE concentration and clinical efficacy could be assessed.

Additional studies would investigate alternative dosing schedules and dosing

intervals and compare anti-IgE treatment to placebo formulations. The efficacy of

topically applied anti-IgE would also be studied. The pharmaceutical composition

295 of the invention, administered by any acceptable route, is expected to have a

substantial beneficial effect for patients suffering from atopic dermatitis.

The foregoing description, terms, expressions and examples are exemplary

only and not limiting. The invention includes all equivalents of the foregoing

embodiments, both known and unknown. The invention is limited only by the claims

300 which follow and not by any statement in any other portion of this document or in

any other source.

Claims

16 What is Claimed Is:

1. A pharmaceutical composition for treating atopic dermatitis comprising an IgE

antagonist.

305 2. A pharmaceutical composition for treating atopic dermatitis comprising an anti-

IgE antibody which binds to secreted IgE but not to basophils.

3. A pharmaceutical composition for treating atopic dermatitis comprising an anti-

IgE antibody which binds to secreted IgE and membrane-bound IgE but not to

basophils.

310 4. A pharmaceutical composition for treating atopic dermatitis comprising an anti-

IgE antibody which binds to secreted IgE and membrane-bound IgE but not to

basophils and not to IgE which is bound to the FcεRII receptor.

5. The pharmaceutical composition of any of claims 2 to 4 wherein the anti-IgE

antibody does not bind to mast cells.

315 6. The pharmaceutical composition of any of claims 2 to 4 wherein the anti-IgE

antibody is a monoclonal antibody.

7. The pharmaceutical composition of claim 6 wherein the anti-IgE antibody is a

chimeric, humanized (CDR-grafted), or human antibody.

8. The pharmaceutical composition of claim 6 wherein the anti-IgE antibody targets

320 human IgE,

9. The pharmaceutical composition of claim 8 wherein the anti-IgE antibody has a

human lgG1 or lgG3 heavy chain constant region.

10. The pharmaceutical composition of any of claims 2 to 9 further including 17 excipients and solvents to make the composition suitable for internal use.

325 11. The pharmaceutical composition of claim 10 wherein the excipients and

solvents make it suitable for subcutaneous or intravenous injection.

12. A pharmaceutical composition for treating atopic dermatitis comprising an anti-

IgE antibody with the same properties as that produced by the cell line on deposit

at the American Type Culture Collection under Accession Number BRL 10706.

330 13. A pharmaceutical composition for treating atopic dermatitis comprising an anti-

IgE antibody with the same structure as that produced by the cell line on deposit at

the American Type Culture Collection under Accession Number 11131.