U.S. patent application number 11/046159 was filed with the patent office on 2005-08-18 for polyclonal antibody composition for treating allergy.
This patent application is currently assigned to Symphogen A/S. Invention is credited to Drejer, Kirsten, Haurum, John S., Morch, Ulrik Gregers Winther.
Application Number | 20050180967 11/046159 |
Document ID | / |
Family ID | 26906647 |
Filed Date | 2005-08-18 |
United States Patent
Application |
20050180967 |
Kind Code |
A1 |
Haurum, John S. ; et
al. |
August 18, 2005 |
Polyclonal antibody composition for treating allergy
Abstract
A pharmaceutical composition for treating allergy is described.
The composition comprises as an active ingredient a recombinant
polyclonal antibody or a mixture of different monoclonal antibodies
capable of reacting with or binding to an allergen together with
one or more pharmaceutically acceptable excipients. The composition
may be used topically as a solution, dispersion, powder, or in the
form of microspheres. The polyclonal antibody is preferably a
recombinant polyclonal antibody produced by phage display
technology. The pairing of specific immunoglobulin variable region
light chain and heavy chain maintained from the original polyclonal
immune response or selected by panning using the allergen in
question is preferably maintained by bulk transfer of the pairs
into an expression vector.
Inventors: |
Haurum, John S.;
(Copenhagen, DK) ; Drejer, Kirsten; (Vaerlose,
DK) ; Morch, Ulrik Gregers Winther; (Frederiksberg,
DK) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Assignee: |
Symphogen A/S
Lyngby
DK
|
Family ID: |
26906647 |
Appl. No.: |
11/046159 |
Filed: |
January 28, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11046159 |
Jan 28, 2005 |
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09866573 |
May 25, 2001 |
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6849259 |
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60211981 |
Jun 16, 2000 |
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Current U.S.
Class: |
424/130.1 |
Current CPC
Class: |
A61K 2039/541 20130101;
C07K 16/18 20130101; A61K 2039/505 20130101; C07K 16/14 20130101;
C07K 16/16 20130101; A61K 2039/545 20130101 |
Class at
Publication: |
424/130.1 |
International
Class: |
A61K 039/395 |
Claims
What is claimed is:
1. A pharmaceutical composition comprising as an active ingredient
a recombinant polyclonal antibody or a mixture of individual
monoclonal antibodies or an isolated or purified polyclonal
antibody capable of reacting with or binding to an allergen
together with one or more pharmaceutically acceptable
excipients.
2. The pharmaceutical composition of claim 1, wherein the active
ingredient is a recombinant polyclonal antibody.
3. The pharmaceutical composition of claim 1, wherein the active
ingredient is a mixture of individual monoclonal antibodies.
4. The pharmaceutical composition of claim 1, wherein the active
ingredient is an isolated or purified polyclonal antibody.
5. The pharmaceutical composition of claim 1, where the composition
is free of the allergen to which the antibody is reactive or
binds.
6. The pharmaceutical composition of claim 1, wherein the
composition comprises at least one pharmaceutically acceptable
excipient capable of effecting topical application of said
recombinant polyclonal antibody or said mixture of individual
monoclonal antibodies or said isolated or purified polyclonal
antibody.
7. The pharmaceutical composition of claim 5, wherein said
composition is intended for topical administration to the
oropharynx, nasal cavity, respiratory tract, gastrointestinal
tract, conjunctival mucosa, vagina, urogenital mucosa, or for
dermal application.
8. The pharmaceutical composition of claim 7, wherein the
respiratory tract comprises the nasal, oral, pharyngeal, bronchial
or alveolar mucosa.
9. The pharmaceutical composition of claim 1, wherein said
composition is provided as a solution, dispersion, powder, or in
the form of microspheres.
10. The pharmaceutical composition of claim 2, wherein the
recombinant polyclonal antibody is generated by phage display
technology.
11. The pharmaceutical composition of claim 10, wherein the
recombinant polyclonal antibody is generated under such conditions
that the immunoglobulin heavy chain variable region and light chain
variable region gene segments are linked together in a parental
library in order to allow for the bulk transfer of variable region
light chain and heavy chain gene pairs from one vector to another,
while allowing stable pairing of specific immunoglobulin variable
region light chain and heavy chain gene segments as they are
present upon selection from the parental library of immunoglobulin
variable region light chain and heavy chain gene segment pairs
encoding antibody molecules capable of reacting with or binding to
an allergen.
12. The pharmaceutical composition of claim 10, wherein the
recombinant polyclonal antibody is generated under such conditions
that the immunoglobulin heavy chain variable region and light chain
variable region gene segments are linked together in order to allow
for the bulk transfer of specific variable region light chain and
heavy chain gene pairs from one vector to another, while allowing
stable pairing of specific immunoglobulin variable region light
chain and heavy chain gene segments as they are present in the
original polyclonal immune response of an animal or human
individual.
13. The pharmaceutical composition of claim 1, wherein the allergen
is an allergen of a house dust mite, dander from a cat, a dog, or a
horse, tree pollen, grass pollen, or fungi.
14. The pharmaceutical composition of claim 1, wherein the
recombinant polyclonal antibody or the mixture of monoclonal
antibodies or the isolated or purified polyclonal antibody is
provided in an amount in the range of 1 .mu.g to 1 g per unit
dosage form.
15. The pharmaceutical composition of claim 14, wherein the
recombinant polyclonal antibody or the mixture of monoclonal
antibodies or the isolated or purified polyclonal antibody is
provided in an amount in the range of 1 .mu.g to 1000 .mu.g per
unit dosage form.
16. A method of preventing or treating allergy, said method
comprising administering to a patient in need thereof a
pharmaceutical composition comprising as an active ingredient a
recombinant polyclonal antibody or a mixture of individual
monoclonal antibodies or an isolated or purified polyclonal
antibody capable of reacting with or binding to an allergen to
which the patient has shown or is predisposed to develop an
allergic reactions together with one or more pharmaceutically
acceptable excipients.
17. A method of inducing tolerance to an allergen, said method
comprising administering to a patient, who if untreated would be
likely to show allergic reaction to the allergen, a composition
comprising as an active ingredient a recombinant polyclonal
antibody or a mixture of individual monoclonal antibodies or an
isolated or purified polyclonal antibody capable of reacting with
or binding to an allergen and inducing tolerance to the allergen in
the patient.
Description
FIELD OF INVENTION
[0001] The present invention relates to a composition comprising a
recombinant polyclonal antibody or a mixture of different
monoclonal antibodies or an isolated or purified polyclonal
antibody capable of reacting with or binding to an allergen, as
well as the use of a polyclonal antibody capable of reacting with
or binding to an allergen for the treatment of allergy.
BACKGROUND OF THE INVENTION
[0002] The protective effects of humoral immunity are known to be
mediated by a family of structurally related glycoproteins called
antibodies. Antibodies initiate their biological activity by
binding to antigens. Antibody binding to antigens is generally
specific for one antigen and the binding is usually of high
affinity. Antibodies are produced by B-lymphocytes. Blood contains
many different antibodies, each derived from a clone of B-cells and
each having a distinct structure and specificity for antigen.
Antibodies are present on the surface of B-lymphocytes, in the
plasma, in interstitial fluid of the tissues and in secretory
fluids such as saliva and mucus on mucosal surfaces.
[0003] All antibodies are similar in their overall structure,
accounting for certain similarities in physiochemical features such
as charge and solubility. All antibodies have a common core
structure of two identical light chains, each about 24 kilodaltons,
and two identical heavy chains of about 55-70 kilodaltons each. One
light chain is attached to each heavy chain, and the two heavy
chains are attached to each other. Both the light and heavy chains
contain a series of repeating homologous units, each of about 110
amino acid residues in length which fold independently in a common
globular motif, called an immunoglobulin (Ig) domain. The region of
an antibody molecule formed by the association of the two heavy
chains is hydrophobic. Antibodies are known to cleave at the site
where the light chain attaches to the heavy chain when they are
subjected to adverse physical or chemical conditions. Because
antibodies contain numerous cysteine residues, they have many
cysteine-cysteine disulfide bonds. All Ig domains contain two
layers of beta-pleated sheets with three or four strands of
anti-parallel polypeptide chains.
[0004] Despite their overall similarity, antibody molecules can be
divided into distinct classes and subclasses based on
physiochemical characteristics such as size, charge and solubility,
and on their behavior in binding to antigens. In humans, the
classes of antibody molecules are: IgA, IgD, IgE, IgG and IgM.
Members of each class are said to be of the same isotype. IgA and
IgG isotypes are further sub-divided into subtypes called
IgA.sub.1, IgA.sub.2 and IgG.sub.1, IgG.sub.2, IgG.sub.3 and
IgG.sub.4. The heavy chains of all antibody molecules in an isotype
share extensive regions of amino acid sequence identity, but differ
from antibodies belonging to other isotypes or subtypes. Heavy
chains are designated by the letters of the Greek alphabet
corresponding to the overall isotype of the antibody molecule,
e.g., IgA contains .alpha., IgD contains .delta., IgE contains
.epsilon., IgG contains .gamma., and IgM contains .mu. heavy
chains. IgG, IgE and IgD circulate as monomers. IgA molecules
secreted through the epithelia into the mucosal lining of body
cavities are homodimers whereas IgM molecules are pentamers.
Circulating IgA exists mainly as a monomer. Multimeric forms of IgA
and IgM are both stabilized by the so called J chain. Secreted IgA
(S-IgA) is produced by B cells residing in lamina propria and taken
up by epithelial cells on the basolateral side through the
poly-immunoglobulin receptor (pIgR), transported through the
epithelial cell and secreted into the mucosa on the luminal side.
When the IgA:J chain:pIgR complex is released, the pIgR is cleaved
by a protease and a part of the pIgR molecule called the secretory
component (SC) remains bound to the IgA:J chain complex. Thus,
S-IgA is a complex consisting of IgA, the J chain, and the SC of
which the two latter are covalently bound to the IgA molecule
through disulphide bonds. S-IgA is very resistant to the
proteolytic environment of the epithelial mucosa e.g. in the
respiratory or the gastrointestinal tract, and as such make up the
primary specific immune system in these sites. It has been
demonstrated that S-IgA has an immunomodulating effect and may
induce tolerance to the antigens they bind.
[0005] There are between 10.sup.8 and 10.sup.10 structurally
different antibody molecules in every individual, each with a
unique amino acid sequence in their antigen combining sites.
Sequence diversity in antibodies is predominantly found in three
short stretches within the amino terminal domains of the heavy and
light chains called variable (V) regions, to distinguish them from
the more conserved constant (C) regions.
[0006] Immunoglobulin E (IgE) is responsible for so-called type 1
hypersensitivity which manifest itself as common diseases such as
allergic rhinitis, allergic conjunctivitis, hay fever, allergic
(extrinsic) asthma, bee venom allergy, and food allergy.
Allergen-specific IgE is produced in excess in patients with
IgE-mediated allergies. IgE circulate in the blood and bind to
high-affinity Fc receptors for IgE on basophils and mast cells in
blood, various tissues, or on mucosal surfaces. In most allergic
responses, the allergens enter the body of a patient through
inhalation, ingestion, or through the skin. The allergen molecules
bind to preformed IgE already bound to the high-affinity receptor
Fc.epsilon.RI on the surfaces of mast cells and basophils,
resulting in the crosslinking of several IgE molecules and
triggering the release of histamine and other inflammatory
mediators causing the various allergic symptoms.
[0007] Among the tissues that are most susceptible to local
IgE-mediated allergic reactions are the conjunctiva, the mucosa of
the nasal cavity or the oropharynx (allergic rhinitis), the mucosal
linings of the bronchial tract, and the gastrointestinal mucosa.
Thus, allergens enter the respiratory tract through inhalation and
get trapped on the mucosal surfaces of the nasal lining or the
bronchial passages of the respiratory tract. Airborne allergens
also get in contact with moist surfaces of eyes and ears and are
retained on the mucosa. The mucosal tissues are densely populated
with mast cells and allergens arriving at these sites may therefore
bind IgE and activate mast cells.
[0008] The therapeutic principles and treatment modalities in the
management of allergy have not changed substantially in recent
years. Immunosuppressive drugs such as steroids for suppressing
immune activities and bronchial dilators for relieving asthma
symptoms have long been the main treatment modality for patients
with allergic asthma. Desensitization immunotherapy is the most
important novel therapy for severely affected patients, but the
medical advances have been limited to refining the classification
of the allergenic substances, improving diagnostic methods, and
providing a better controlled and broader library of allergen
extracts for immunotherapy. As for research, progress has been made
in the identification and isolation of major allergenic components
of allergenic substances. For example, the major allergic
components of ragweed, house dust mites, and cat and dog dander and
saliva have been identified. When the allergen particles, e.g.
timothy grass pollen arrive to the airway mucosa they disintegrate
into major and minor allergic components.
[0009] Antibodies have been suggested for a number of clinical
treatments: MedImmune Inc. is studying the use of humanized
anti-respiratory syncytial virus (RSV) monoclonal antibodies and
markets a polyclonal anti-RSV human immunoglobulin product
(RespiGam) isolated from human donor blood and used to treat RSV
infection. MedImmune also markets CytoGam, an anti-CMV
(cytomegalovirus) human immunoglobulin for the treatment of CMV
infection. IDEC and Genentech are jointly performing clinical
trials of a chimeric mouse-human monoclonal antibody (Rituximab)
aimed at the CD20 antigen found on mature B cells and most
non-Hodgkin's lymphoma tumors for the treatment of relapsed or
refractory low-grade non-Hodgkin's lymphoma. GalaGen is studying
the use of bovine polyclonal immunoglobulin (Diffistat-G) for
treatment of Clostridium difficile antibiotic associated diarrhea.
SmithKline Beecham and Schering-Plough are developing an anti-IL-5
monoclonal antibody which has been shown in clinical trials to
prevent eosinophilic inflammation and airway constriction. An
anti-IgE monoclonal antibody is being developed by Genentech to
"switch-off" allergies. The antibody Rhu-Mab-E25, which is a
humanized chimeric IgG.sub.1 monoclonal antibody specific for a
unique epitope on human high affinity IgE receptors
(Fc.epsilon.RI), has been shown to reduce free IgE levels after the
first administration by injection. It attenuated both early and
late phase responses to inhaled allergens after multiple
injections. Examples of antibodies used therapeutically also
include a nebulized IgG (Sandoz), which is used intranasally
against RSV; HNK20 (Oravax), an anti-RSV IgA; and 4B9 (Bristol
Myers-Squibb), an anti-group B Streptococcus IgM monoclonal
antibody. Other therapeutically useful monoclonal antibodies
include monoclonal anti-CD4 antibodies, anti-IL-2 antibodies and
anti-IL-4 antibodies.
[0010] The immunotherapy of RSV infection using small particle
aerosols of IgG has been disclosed by Piazza et al. (J. Infect.
Dis., Vol. 166, pp. 1422-1424, 1992). In this study it was shown
that a 15-minute exposure to an aerosolized 5% solution of IgG
effected a 50-fold reduction in pulmonary virus. Brown (Aerosol
Science and Technology, Vol. 24, pp. 45-56, 1996) discloses the use
of antibodies as in hibitors or antagonists of cytokines to depress
respiratory inflammatory diseases or allergen-induced asthmatic
responses. Also mentioned is local respiratory delivery of
pathogen-specific antibody for treatment of acute viral or
bacterial respiratory infections.
[0011] Antibody liposomes, i.e., immunoliposomes, are disclosed by
Maruyama et al. (Biochim. Biophys. Acta, Vol. 1234, pp. 74-80,
1995). Coating liposomes with antibody leads to enhanced uptake of
the liposome by the reticuloendothelial system. Human monoclonal
antibodies are known to be useful as anti-tumor agents. A
mouse/human monoclonal IgG antibody specific for the Lewis Y
antigen found on the surface of tumor cells is disclosed by Paborji
et al. (Pharmaceutical Research, Vol. 11, No. 5, pp. 764-771,
1994). The use of antibodies in metered-dose propellant driven
aerosols for passive antibody aerosol therapy against respiratory
infections is suggested in Brown et al. (Journal of Immunological
Methods, Vol. 176, pp. 203-212, 1994). Immune responses in the
respiratory tract are of great importance for protection against
infections of the respiratory system and for their involvement in
respiratory allergies and asthma. Effective targeting of
immunomodulating reagents including monoclonal antibodies to the
respiratory tract is shown to be of benefit in increasing local
immunity to respiratory pathogens or decreasing immune-mediated
respiratory pathology. Inhaled immunoconjugates, immunoliposomes or
immunomicrospheres have application in the lung as killers of
cancer cells (immunoconjugates) or, in the case of immunoliposomes
and microspheres, as stealth delivery particles of a variety of
therapeutic agents. An IgM anti-group B Streptococcus monoclonal
antibody is disclosed by Gombotz et al. (Pharmaceutical Research,
Vol. 11, pp. 624-632, 1994).
[0012] U.S. Pat. No. 5,670,626 proposes the use of monoclonal
antibodies for the treatment of IgE-mediated allergic diseases such
as allergic rhinitis, allergic asthma and allergic conjunctivitis
by employing monoclonal antibodies to inhibit the entry of
allergenic molecules into mucosal tissues. The binding of
allergenic molecules by antibodies is assumed to inhibit the
allergens from being taken up by mucosal epithelial cells.
[0013] In certain clinical situations, the use of monoclonal
antibodies is associated with specific disadvantages. Thus,
monoclonal antibodies are directed against single antigenic
epitopes. Therefore, if the target is of a complex nature
presenting many different epitopes then the functional avidity of
the monoclonal antibody may be low or lowered below a critical
threshold allowing the target to escape elimination through immune
recognition.
[0014] Also, because monoclonal antibodies are directed against
single antigenic determinants, the density of the antibody targets
on e.g. allergens may not be high enough to mediate elimination of
the allergen. The efficient activation of complement similarly
requires high target antibody densities which may not be achieved
with single specificity monoclonal antibodies.
[0015] Thus, in the case of allergens, monoclonal antibodies are
sub-optimal as they are directed against single epitopes. The
majority of allergens are complex proteins, consisting of many
protein and peptide epitopes, and existing in many variants. Thus,
a single monoclonal antibody preparation cannot be expected to
exhaustively cover more than a minority of the possible epitopes on
an allergen, e.g. a pollen particle or proteins from cat dander.
This means that if the desired clinical effect of an antibody can
be characterized as a complete blocking of the available antibody
epitopes, then a single monoclonal antibody will not be sufficient.
Further, if an antibody preparation should preferably be developed
against several homologous allergens from closely related
allergens, e.g. pollens, or against several proteins from one
allergen source e.g. animal dander, then a single monoclonal
antibody will not meet the required efficacy.
[0016] Nevertheless, a paper by Schwarze and coworkers (Am. J.
Resp. Crit. Care Med., Vol. 158, pp. 519-525, 1998) investigated
the therapeutic efficacy of a monoclonal antibody directed against
the major ragweed allergen Amb a/in a murine allergy model based on
mice (Balb/c) sensitized and challenged with both Amb a/and whole
ragweed extracts. It was demonstrated that administration of the
monoclonal IgA antibody before allergen exposure decreased airway
responsiveness to metacholine challenge, and decreased the number
of pulmonary eosinophils and Amb a/-specific IgE levels in serum.
Moreover, the study indicate that administration of IgA had an
immunomodulatory effect implying that IgA treatment could have a
long-term desensitizing effect on allergy. However, it must be
stressed that this allergen model is based on the induction of
allergy-like symptoms using a single allergen, Amb a I. Thus, the
study does not take into account that the vast majority of
allergies are caused by reactions towards a number of allergen
proteins and epitopes derived from a single allergen particle,
which emphasizes the need for a polyclonal antibody mixture in this
regime of treatment. Furthermore, human allergy is profoundly more
complex than the allergy-like symptoms induced in an inbred mouse
strain (Inhal. Toxicol., Vol 12, pp. 829-622, 2000). Consequently,
the potential usefulness of monoclonal antibodies as allergen
blocking agents is limited. Finally, monoclonal antibodies may
display cross-reactivity to antigenic structures of host cell
tissue resulting in potential unwanted side effects. When this
occurs the cross-reactivity cannot be removed by adsorption.
Therefore a large number of different monoclonal antibodies may
need to be produced in order to generate the desired combination of
antigen specificity and target selectivity, and even so there still
remains a significant risk of cross-reactivity towards endogenous
self-antigens in a proportion of patients.
[0017] A separate issue is the generation of human anti-mouse
antibody responses (HAMA). Conventional murine monoclonal
antibodies are foreign proteins to the human recipient, and
therefore a HAMA immune response is often elicited in the
recipient, which may lead to unwanted side effects in addition to
reduced treatment efficacy. In order to circumvent this problem,
chimeric monoclonal antibodies possessing human constant (C)
regions and murine variable (V) regions have been developed.
Furthermore humanized monoclonal antibodies, where only the
hypervariable complementarity determining region (CDR) is derived
from mouse monoclonal antibodies and finally, so-called fully human
monoclonal antibodies produced in mice transgenic for human
immunoglobulin genes have been developed to avoid these problems.
However, a potential for the generation of anti-idiotype antibody
responses specific for the V-region specificity determining CDR
still exists when injecting large amounts of monoclonal antibodies
with identical V-regions.
[0018] For these reasons as outlined above, it may often be
preferable to use polyclonal antibodies.
[0019] In WO 98/10776 it is theorized that phospholipase A.sub.2
(PLA.sub.2) is involved in the pathogenesis of many diseases acting
as an inflammatory mediator promoting chronic inflammation. Thus it
is suggested to use serum reactive with at least one phospholipase
A.sub.2 enzyme for the treatment of neoplasms in mammals. There is
no suggestion to use polyclonal antibodies for blocking the uptake
of an allergen by topical administration of an antibody binding to
the allergen.
[0020] U.S. Pat. No. 4,740,371 describes a modification of allergen
immunotherapy whereby an immune complex of the allergen and an
antibody thereto is used for desensitization treatment, the
antibody being present in molar excess with respect to the allergen
to prevent an anaphylactic response. The purpose of the inclusion
of the antibody in this treatment is to decrease the risk of
allergic side effects such as anaphylactic shock to the
desensitization treatment. The proportion of antibody to be added
to the allergen is defined essentially by the neutralizing power of
the antibody. Enough antibody must be used so that when the
composition is administered, there is practically no allergic
effect induced by the allergen. The adding of antibody to the
allergen composition is solely a remedy to avoid side effects of
the allergen exposure, the treatment still being an allergen
immunotherapy.
[0021] There are several drawbacks of using conventional polyclonal
antibodies in the treatment of allergy. First of all, polyclonal
antibodies in the form of IgG purified from hyperimmune human serum
is available in limited supply and in amounts insufficient for the
treatment of allergic diseases and other common conditions. Also,
gamma globulin preparations are expensive to produce, and display
low efficacy due to their mixed nature containing an overwhelming
majority of non-specific human serum immunoglobulin reactivities.
Also, there exist a real risk of transmitting contaminating
reagents, including infectious microorganisms (hepatitis virus,
HIV, prions, others), or mitogens, cytokines and toxins. Finally,
the variability between preparations remains a major problem. In
order to solve the problem of supply, xenogeneic sources of
polyclonal antibodies including serum from immunized non-human
animals have been tested. However, such compositions may result in
the generation of potent anti-xenoantibody responses, and carries a
real risk of serious side effects such as anaphylactic shock or
serum sickness, as well as the transmission of xenotropic
infections.
[0022] U.S. Pat. No. 5,789,208 describes the use of a recombinant
polyclonal antibody for vaccine therapy and prophylaxis to treat or
prevent neoplastic diseases. The antibodies are used for boosting a
patient's immune system for the possible later recognition of the
antigen to which the antibody binds and thereby initiate an
elimination reaction. The vaccination will have to be repeated to
be effective. There is no suggestion to use polyclonal antibodies
reacting with or binding to allergens in allergy treatment where
the polyclonal antibodies should be administered completely
differently before, during, or shortly after the patient has been
exposed to an allergen.
SUMMARY OF THE INVENTION
[0023] Accordingly, the present invention relates to a
pharmaceutical composition comprising as an active ingredient a
recombinant polyclonal antibody or a mixture of different
monoclonal antibodies or an isolated or purified polyclonal
antibody capable of reacting with or binding to an allergen
together with one or more pharmaceutically acceptable
excipients.
[0024] In most embodiments, the pharmaceutical composition
according to the invention is free of the allergen to which the
recombinant polyclonal antibody or the mixture of different
monoclonal antibodies or the isolated or purified polyclonal
antibody bind or is reactive with. However, in special cases during
so-called specific allergen immunotherapy to induce allergen
tolerance in a patient, the allergen-specific polyclonal antibody
may be administered to the patient in conjunction with an allergen
preparation, in order to enable or enhance the efficacy of the
tolerance induction procedure.
[0025] In another aspect, the invention relates to the use of a
polyclonal antibody with desired specificities for the manufacture
of a pharmaceutical composition for the prophylaxis or treatment of
allergy or allergic conditions such as allergic rhinitis, allergic
conjunctivitis, hay fever, asthma, etc.
[0026] In a further aspect, the invention relates to a method of
preventing or treating allergy, which comprises administering to a
patient in need thereof a sufficient amount of a polyclonal
antibody capable of reacting with or binding to an allergen to
which the patient has shown an allergic reaction.
[0027] The use of a polyclonal antibody has potential clinical
advantages compared with the use of a monoclonal antibody due to
the presence of multiple reactivities in a polyclonal antibody
against the allergen-target in question. There may be generated a
polyclonal antibody which has reactivities against all epitopes on
a complex allergen target. Due to the polyclonal nature of the
composition, containing many epitope specificities, the functional
antibody density which can be achieved on complex allergen antigens
when using a polyclonal antibody is significantly higher, than with
a monoclonal antibody. This results in more efficient blocking or
clearance of the target allergen. Further, the polyclonal nature of
the composition enables recognition of and blocking of epitopes on
related, homologous allergen isotypes, due to broad reactivity with
several epitopes shared in part between related allergens,
something which is not enabled by a monoclonal antibody.
[0028] Further, it can be expected that treatment with
allergen-specific polyclonal antibodies of the IgA or IgG isotype
will have an immunomodulating effect by inducing tolerance to an
allergen, and thus have a long term effect in curing the allergy or
reducing the need for further treatment. Thus, a further aspect of
the invention relates to the use of a pharmaceutical composition
according to the invention for prophylactic treatment inducing
tolerance to the allergen. This may even be used in patients where
an allergic reaction has not yet been observed but which patients
due to family history or genetic analysis are likely to develop
allergy to an allergen.
[0029] Furthermore, contrary to a monoclonal antibody, a polyclonal
antibody preparation comprises a mixture of specificities, and
therefore any single and individual, cross-reacting specificity
idiotype will be delivered at a very low concentration, thus
reducing significantly the potential for harmful side-effects, due
to cross-reactivity. In other words, the potential for deleterious
side-effects due to unwanted tissue cross-reactivity is diluted out
in the polyclonal antibody reagent. Further, any unwanted
cross-reactivity of the polyclonal antibody preparation can be
removed by adsorption. If a monoclonal antibody results in an
unwanted cross-reactivity, it is inherent to the single antibody
present and can of course not be removed without destroying the
activity of the preparation.
[0030] Also, in analogy with the properties of polyclonal
antibodies in terms of the diminished potential for
cross-reactivity, polyclonal antibodies will also be much less
likely than monoclonal antibodies to induce a neutralizing
anti-idiotype immune response, since each single epitope-specific
idiotype of the administered polyclonal antibody preparation is
present in a very low quantity or concentration, being below the
threshold for generation of an anti-idiotype response.
[0031] Some of the drawbacks of using conventional polyclonal
antibodies in the form of IgG purified from hyperimmune human
(limited supply, expensive to produce) or serum from normal animals
(anti-xeno-antibody responses, anaphylactic shock) is the use of
serum or other biological material from animals transgenic for
human immunoglobulin genes. Thus, such animals can be immunized
with allergens, and used as a source to isolate allergen-specific
polyclonal antibody products of fully human sequence.
[0032] The immune complexes on the nasal linings will be cleared as
the mucous excretion is swallowed. The immune complexes on the
mucosal surfaces of the tracheal and bronchial airways will be
expelled into the mouth, mixed with saliva, swallowed and digested
in the gastrointestinal tract. In order to achieve betters effects
in adsorbing and clearing allergenic molecules from the mucous
fluids on the mucosal surfaces and preventing any uptake of the
complexed allergen by the mucosal epithelial cells, the
allergen-specific antibody can be conjugated to polymer backbones
or microbeads forming microspheres.
[0033] Thus the pharmaceutical composition according to the
invention may be formulated as a solution, dispersion, powder, or
in form of microspheres.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The term "antibody molecule" describes the single antibody
protein molecule or fragments thereof containing one or more
variable antigen binding domain(s) and constant regions. An
antibody molecule is usually monospecific, but may also be
described as idiospecific, heterospecific, polyspecific or of
unwanted specificity. It cannot be non-specific except in the sense
of non-immunochemical binding. Antibody molecules bind by means of
specific binding sites to specific antigenic determinants or
epitopes on antigens.
[0035] Collectively, antibodies may exist as a population of
molecules where a fraction or all of the members are capable of
reacting with a specific antigen determinant. Thus, in the present
context, the term "antibody" refers to
compositions/mixtures/populations of antibody molecules, such as
they are found as the functional component of anti-serum or immune
serum derived from mammals, or as they are found in monoclonal or
polyclonal antibody compositions with similar functionality
prepared either from human or animal sources or by recombinant
technologies, including transgenic animals and phage display or by
conventional hybridoma technology.
[0036] The term "polyclonal antibody" denotes a mixture of
different antibody molecules which react with more than one
immunogenic determinant of an antigen.
[0037] In the present context, the term "polyclonal antibody"
encompasses a polyclonal antibody isolated or purified from
mammalian blood, secretions, or other fluids, or from eggs, as well
as a mixture of different monoclonal antibodies, and finally a
polyclonal antibody may be produced as a recombinant polyclonal
antibody.
[0038] The term "recombinant polyclonal antibody" refers to a
polyclonal antibody generated by the use of recombinant
technologies, and such polyclonal antibodies are hereafter named
symphobodies. Thus, a symphobody contains a high concentration of
different antibody molecules, all or a majority of which are
displaying a desired binding activity towards an antigen composed
of more than one epitope.
[0039] Symphobodies can be generated by recombinant DNA techniques
followed by expression in eukaryotic cells, including yeast, fungi,
insect, plant, or mammalian cells, or in prokaryotic cells such as
bacteria, or as expressed from virus vectors, or through gene
therapy, or from expression of transgenes in animals.
[0040] Preferably at least 85% of the antibody molecules in the
symphobody preparation are target-specific, more preferably at
least 90% are target-specific, even more preferably at least 95%
are target-specific, and most preferably all antibody molecules in
the symphobody preparation are target-specific.
[0041] By the term "a mixture of different monoclonal antibodies"
is meant a mixture of two or more different monoclonal antibodies.
The term "two or more" in the present context denotes from 2 to
100, preferably from 3 to 60, more preferably from 5 to 40, most
preferably from 10 to 25 different monoclonal antibodies.
[0042] By the term "an isolated or purified polyclonal antibody" is
meant a polyclonal antibody isolated or purified from mammalian
blood, secretions, or other fluids, or from eggs.
[0043] It is to be understood that the expressions "an antibody, a
polyclonal antibody, a recombinant antibody, a mixture of different
monoclonal antibodies and an isolated or purified polyclonal
antibody" all also encompasses functional fragments of the
mentioned antibodies.
[0044] A currently preferred method of preparing a recombinant
polyclonal antibody is by making polyclonal antibody libraries
(PCAL), for instance as disclosed in U.S. Pat. No. 255,789,208 (to
J. Sharon) which is hereby incorporated by reference in its
entirety.
[0045] More specifically, the polyclonal antibody included in the
pharmaceutical composition may be prepared by immunizing an animal,
preferably a mammal, with an allergen of choice followed by the
isolation of antibody-producing B-lymphocytes from blood, bone
marrow, lymph nodes, or spleen. Alternatively, antibody-producing
cells may be isolated from an animal and exposed to an allergen in
vitro against which antibodies are to be raised. The
antibody-producing cells may then be cultured to obtain a
population of antibody-producing cells, optionally after fusion to
an immortalized cell line such as a myeloma.
[0046] More preferably, as a starting material B-lymphocytes may be
isolated from the tissue of an allergic patient, in order to
generate fully human polyclonal antibodies.
[0047] The present composition may also be generated using suitable
tissue from mice, rats, pigs (swine), sheep, bovine material, or
other animals transgenic for the human immunoglobulin genes, as
starting material in order to generate fully human polyclonal
antibodies.
[0048] Particularly, in the case of mice or other animals
transgenic for the human immunoglobulin genes (e.g. as disclosed in
U.S. Pat. No. 5,939,598), the animals may be immunized to stimulate
the in vivo generation of specific antibodies and antibody
producing cells before preparation of the polyclonal antibody
composition from the animal by extraction of B lymphocytes or
purification of polyclonal serum.
[0049] A combinatorial library may be prepared from immunized B
lymphocytes by associating V.sub.L and V.sub.H randomly in a
cloning vector. Thus, the recombinant polyclonal antibody is
generated under such conditions that the immunoglobulin heavy chain
variable region and light chain variable region gene segments are
linked together randomly in order to allow for the bulk transfer of
variable region light chain and heavy chain gene pairs from one
vector to another, while allowing stable pairing of specific
immunoglobulin variable region light chain and heavy chain gene
segments as they are present upon selection from a parental library
of immunoglobulin variable region light chain and heavy chain gene
segment pairs encoding antibody molecules capable of reacting with
or binding to an allergen.
[0050] Single cell PCR may be used in an attempt to retain the
native pairing of V.sub.L and V.sub.H in the single cell. In this
case antibody-producing B-lymphocytes which have been isolated from
animals or humans may be fixed with a fixative solution or a
solution containing a chemical such as formaldehyde, glutaraldehyde
or the like.
[0051] The cells are then permeabilized with a permeabilization
solution comprising for example a detergent such as Brij, Tween,
polysorbate, Triton X-100, or the like. The fixing and
permeabilization process should provide sufficient porosity to
allow entrance of enzymes, nucleotides and other reagents into the
cells without undue destruction of cellular compartments or nucleic
acids therein. Addition of enzymes and nucleotides may then enter
the cells to reverse transcribe cellular V.sub.H and V.sub.L mRNA
into the corresponding cDNA sequences.
[0052] Reverse transcription may be performed in a single step or
optionally together with a PCR procedure, using a reverse
transcriptase, sufficient quantities of the four dNTPs and primers
that bind to the mRNA providing a 3' hydroxyl group for reverse
transcriptase to initiate polymerization. Any primer complementary
to the mRNA may be used, but it is preferred to use primers
complementary to the 3'-terminal end of the V.sub.H and V.sub.L
molecules so as to facilitate selection of variable region
mRNA.
[0053] Upon reverse transcription, the resulting cDNA sequences may
be amplified by PCR using primers specific for immunoglobulin genes
and, in particular, for the terminal regions of the V.sub.H and
V.sub.L nucleic acids. PCR procedures may be followed as disclosed
in, e.g., U.S. Pat. No. 4,683,195. Preferably, the cDNAs are PCR
amplified and linked in the same reaction, using, in addition to
the cDNA primers, one primer for the 5' end of the V.sub.H region
gene and another for the 5' end of the V.sub.L gene. These primers
also contain complementary tails of extra sequence, to allow the
self-assembly of the V.sub.H and V.sub.L genes. After PCR
amplification and linking, the chance of getting mixed products, in
other words, mixed variable regions, is minimal because the
amplification and linking reactions were performed within each
cell. The risk of mixing can be further decreased by utilizing
bulky reagents such as digoxigenin labeled nucleotides to further
ensure that V region cDNA pairs do not leave the cellular
compartment and intermix, but remain within the cell for PCR
amplification and linking. The amplified sequences are linked by
hybridization of complementary terminal sequences. After linking,
sequences may be recovered from cells. For example, after linking,
cells can be washed in a solution of sodium dodecyl sulfate (SDS).
The SDS precipitates out of the cells after incubation on ice and
the supernatant can be electrophoresed into an agarose or
acrylamide gel. Alternatively, or in combination with the SDS
process, using a reagent such as digoxigenin-linked nucleotides,
DNA products synthesized will remain within the cell and be
amplified. The linked product is recovered upon electrophoresis of
the supernatant.
[0054] After electrophoresis of the supernatant, the gel slice
corresponding to the appropriate molecular weight of the linked
product is removed and the DNA isolated on, for example, silica
beads. The recovered DNA can be PCR amplified using terminal
primers, if necessary, and cloned into vectors which may be
plasmids, phages, cosmids, phagemids, viral vectors or combinations
thereof. Convenient restriction enzyme sites may be incorporated
into the hybridized sequences to facilitate cloning. These vectors
may also be saved as a library of linked variable regions for later
use.
[0055] The linked V.sub.H and V.sub.L region genes may be PCR
amplified a second time using terminal nested primers, yielding a
population of DNA fragments which encode the linked V.sub.H and
V.sub.L genetic regions. The grouping of V.sub.H and V.sub.L
combinations is an advantage of this process and allows for the in
mass or batch transfer of all clones and all DNA fragments during
this and all cloning procedures.
[0056] Preferably, the recombinant polyclonal antibody may be
generated under such conditions that the immunoglobulin heavy chain
variable region and light chain variable region gene segments are
linked together in a head-to head orientation, in order to allow
for the bulk transfer of variable region light chain and heavy
chain pairs from one vector to another, including from phage to
vector, and including from the cell of origin to phage or vector,
resulting in a stable pairing of specific immunoglobulin variable
region light chain and heavy chains gene segments as they are found
in the original polyclonal immune response of the animal or human
individual.
[0057] It may sometimes be desirable to treat the variable region
gene sequences with a mutating agent. Mutating agents create point
mutations, gaps, deletions or additions in the genetic sequence
which may be general or specific, or random or site directed.
Useful mutating agents include ultraviolet light, gamma
irradiation, chemicals such as ethidium bromide, psoralen and
nucleic acid analogs, or DNA modifying enzymes such as restriction
enzymes, transferases, ligases and specific and nonspecific
nucleases and polymerases. Moreover it may be feasible to use
mutator strains. In particular, random mutations may be introduced
in the CDRs of the V.sub.H and V.sub.L region genes by
oligonucleotide directed mutagenesis. Mutations introduced into the
gene sequence will ultimately increase library complexity and
diversity as well as affinity for antigen which may further
increase the library's usefulness in treatment. Furthermore, such
mutagenesis may be used on a single V.sub.H and V.sub.L pair or on
a defined group of such pairs to generate a library de novo.
[0058] Cloning is performed, for example, by cleaving the cDNA and
vector sequences with a restriction enzyme, if necessary isolating
certain nucleic acid fragments, mixing the fragments together in
the presence of ligase in a suitable balanced salt solution, and
incubating the mixture under enzymatically acceptable conditions
for a prescribed period of time. Using different enzyme recognition
sites at each terminus of the cDNA, cloning orientation can be
predetermined.
[0059] Vectors are transformed into suitable host cells and the
cultures amplified to expand the different populations of vectors
that comprise the library. Host cells for prokaryotic vectors may
be a culture of bacteria such as Escherichia coli. Host cells for
eukaryotic vectors may be a culture of eukaryotic cells such as any
mammalian, insect or yeast cell lines adapted to tissue culture.
Bacterial cells are transformed with vectors by calcium
chloride-heat shock or electroporation, although many other
transformation procedures would also be acceptable. Eukaryotic
cells are transfected with calcium phosphate precipitation or
electroporation, although many other transformation procedures
would also be acceptable. The DNA fragments may be cloned into
prokaryotic or eukaryotic expression vectors, chimeric vectors or
dual vectors. The expression vector may be a plasmid, cosmid,
phage, viral vector, phagemid and combinations thereof, but is
preferably a phage display vector wherein the recombinant product
is expressed on the phage surface to facilitate screening and
selection. Useful transcriptional and translational sites may be
placed on the expression vector including RNA polymerase
recognition regions such as a TATA box site, a CAT site, an
enhancer, appropriate splicing sites, if necessary, a AT rich
terminal region and a transcription initiation site. Useful sites
to facilitate translation include translational start and stop
sites and ribosome binding sites. Typically, some of the more
useful sites for efficient eukaryotic expression, such as the SV40,
CMV, HSV or baculovirus promoter/enhancer region, are derived from
viruses. The resulting recombinant antibody may be of the murine
class IgG.sub.1, IgG.sub.2a, IgG.sub.2b, IgM, IgA, IgD or IgE, the
human classes IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4,
IgA.sub.1, IgA.sub.2, IgA.sub.2, IgD or IgE, or combinations or
fragments thereof. Preferably, the chimeric antibody library is
composed of primarily IgG antibodies or Fab antibody fragments.
[0060] Selection of a recombinant polyclonal antibody with desired
specificity can be performed e.g. by affinity selection (panning)
using an allergen-coated surface for binding the phage particles
exhibiting a relevant antibody specificity. The majority of phages
in the phage library are eliminated by washing and the bound phage
particles are retrieved by harsher conditions (elution). After the
selection procedures, the V.sub.L and V.sub.H antibody gene pairs
in the selected library of phage particles can be subcloned into a
different vector designed for expression of the recombinant
polyclonal antibody as a complete antibody molecule or a fragment
thereof such as a Fab fragment.
[0061] The use of recombinant DNA technology for generating a
recombinant polyclonal antibody is a cost-effective way of
generating antibodies, and the production of well-characterized,
polyclonal antibody preparations with desired specificities, would
overcome the above problems with conventional polyclonal antibody
sera and individual monoclonal antibodies and allow the use of such
reagents for the prophylaxis or treatment of allergy or allergic
conditions, e.g. asthma.
[0062] Pharmaceutical Compositions
[0063] In a preferred embodiment, the pharmaceutical composition of
the invention is one intended for topical
administration/application to mucosa, such as the oropharynx, nasal
cavity, respiratory tract, gastrointestinal tract, eye such as the
conjunctival mucosa, vagina, urogenital mucosa, or for dermal
application.
[0064] A particularly interesting use of the pharmaceutical
composition is for application to the nasal, bronchial or pulmonary
mucosa. Specifically, the topical treatment of allergy using
inhaled polyclonal antibodies would be a particularly useful
application of such reagents, allowing the discovery and
development of novel therapeutic or preventive modalities which are
cheap to produce, harmless and of no toxicity, and aimed towards a
disease afflicting a very large proportion of the human
population.
[0065] In order to obtain optimal delivery of the polyclonal
antibody to the pulmonary cavity in particular, it may be
advantageous to add a surfactant such as a phosphoglyceride, e.g.
phosphatidylcholine, and/or a hydrophilic or hydrophobic complex of
a positively or negatively charged excipient and a charged antibody
of the opposite charge.
[0066] Other excipients suitable for pharmaceutical compositions
intended for delivery of the polyclonal antibody to the respiratory
tract mucosa may be from the group consisting of a) carbohydrates,
e.g., monosaccharides such as fructose, galactose, glucose,
D-mannose, sorbiose, and the like; disaccharides, such as lactose,
trehalose, cellobiose, and the like; cyclodextrins, such as
2-hydroxypropyl-.beta.-c- yclodextrin; and polysaccharides, such as
raffinose, maltodextrins, dextrans, and the like; b) amino acids,
such as glycine, arginine, aspartic acid, glutamic acid, cysteine,
lysine and the like; c) organic salts prepared from organic acids
and bases, such as sodium citrate, sodium ascorbate, magnesium
gluconate, sodium gluconate, tromethamine hydrochloride, and the
like; d) peptides and proteins, such as aspartame, human serum
albumin, gelatin, and the like; e) alditols, such as mannitol,
xylitol, and the like, f) polycationic polymers, such as chitosan
or a chitosan salt or derivative.
[0067] Over the years certain drugs have been sold in compositions
suitable for forming a drug dispersion for oral inhalation
(pulmonary delivery) to treat various conditions in humans. Such
pulmonary drug delivery compositions are designed to be delivered
by inhalation by the patient of the drug dispersion so that the
active drug within the dispersion can reach the lung.
[0068] Pulmonary drug delivery can itself be achieved by different
approaches, including liquid nebulizers, aerosol-based metered-dose
inhalers (MDI's) and dry powdered dispersion devices.
Chlorofluorocarbon (CFC) based MDI's are losing favor because of
their adverse effect on the environment. Dry powder dispersion
devices, which do not rely on CFC aerosol technology, are promising
for delivering drugs that may be readily formulated as dry powders.
Many otherwise labile macromolecules may be stably stored as
lyophilized or spray dried powders, either by themselves or in
combination with suitable powder carriers.
[0069] Many pharmaceutical compositions, including antibodies, are
quite expensive. Thus, the ability to efficiently formulate,
process, package and deliver the dry powders with minimal loss of
drug is critical.
[0070] An important requirement for hand held and other powder
delivery devices is efficiency. It is important that the delivered
dose be relatively high to reduce the number of breaths required to
achieve a total dosage. The ability to achieve both adequate
dispersion and small dispersed volumes is a significant technical
challenge that requires in part that each unit dosage of the powder
composition be readily and reliably dispersible. Certain pulmonary
delivery devices, such as those disclosed in U.S. Pat. No.
5,797,392, U.S. Pat. No. 5,458,135 and International Patent
Publication WO96/09085 are useful for pulmonary delivery of dry
powder drugs. Other administration forms of the present composition
include liquids, gels, ointments or other suitable formulations for
ocular administration, sprays, aerosols, powders, or other
compositions for the administration into the nasal cavity, chewing
gum, pasta or other compositions for oral cavity, creams,
ointments, lotions, gels or other compositions suitable for the
application onto the skin, vagitories, gels or other compositions
suitable for application onto the vaginal or urogenital mucosa or
formulated as capsules or tablets for the administration into the
digestive tract. For dermal application, the polyclonal antibody
may suitably be formulated with one or more of the following
excipients: solvents, buffering agents, preservatives, humectants,
chelating agents, antioxidants, stabilizers, emulsifying agents,
suspending agents, gel-forming agents, ointment bases, penetration
enhancers, perfumes, and skin protective agents.
[0071] Examples of solvents are e.g. water, alcohols, vegetable or
marine oils (e.g. edible oils like almond oil, castor oil, cacao
butter, coconut oil, corn oil, cottonseed oil, linseed oil, olive
oil, palm oil, peanut oil, poppyseed oil, rapeseed oil, sesame oil,
soybean oil, sunflower oil, and tea seed oil), mineral oils, fatty
oils, liquid paraffin, polyethylene glycols, propylene glycols,
glycerol, liquid polyalkylsiloxanes, and mixtures thereof.
[0072] Examples of buffering agents are e.g. citric acid, acetic
acid, tartaric acid, lactic acid, hydrogenphosphoric acid, diethyl
amine etc.
[0073] Suitable examples of preservatives for use in compositions
are parabenes, such as methyl, ethyl, propyl p-hydroxybenzoate,
butylparaben, isobutylparaben, isopropylparaben, potassium sorbate,
sorbic acid, benzoic acid, methyl benzoate, phenoxyethanol,
bronopol, bronidox, MDM hydantoin, iodopropynyl butylcarbamate,
EDTA, benzalconium chloride, and benzylalcohol, or mixtures of
preservatives. Examples of humectants are glycerin, propylene
glycol, sorbitol, lactic acid, urea, and mixtures thereof.
[0074] Examples of antioxidants are butylated hydroxy anisole
(BHA), ascorbic acid and derivatives thereof, tocopherol and
derivatives thereof, cysteine, and mixtures thereof. Examples of
emulsifying agents are naturally occurring gums, e.g. gum acacia or
gum tragacanth; naturally occurring phosphatides, e.g. soybean
lecithin; sorbitan monooleate derivatives; wool fats; wool
alcohols; sorbitan esters; monoglycerides; fatty alcohols;, fatty
acid esters (e.g. triglycerides of fatty acids); and mixtures
thereof.
[0075] Examples of suspending agents are e.g. celluloses and
cellulose derivatives such as, e.g., carboxymethyl cellulose,
hydroxyethylcellulose, hydroxypropylcellulose,
hydroxypropylmethylcellulo- se, carraghenan, acacia gum, arabic
gum, tragacanth, and mixtures thereof.
[0076] Examples of gel bases, viscosity-increasing agents or
components which are able to take up exudate from a wound are:
liquid paraffin, polyethylene, fatty oils, colloidal silica or
aluminum, zinc soaps, glycerol, propylene glycol, tragacanth,
carboxyvinyl polymers, magnesium-aluminum silicates, Carbopol.RTM.,
hydrophilic polymers such as, e.g. starch or cellulose derivatives
such as, e.g., carboxymethylcellulose, hydroxyethylcellulose and
other cellulose derivatives, water-swellable hydrocolloids,
carragenans, hyaluronates (e.g. hyaluronate gel optionally
containing sodium chloride), and alginates including propylene
glycol alginate.
[0077] Examples of ointment bases are e.g. beeswax, paraffin,
cetanol, cetyl palmitate, vegetable oils, sorbitan esters of fatty
acids (Span), polyethylene glycols, and condensation products
between sorbitan esters of fatty acids and ethylene oxide, e.g.
polyoxyethylene sorbitan monooleate (Tween).
[0078] Examples of hydrophobic or water-emulsifying ointment bases
are paraffins, vegetable oils, animal fats, synthetic glycerides,
waxes, lanolin, and liquid polyalkylsiloxanes. Examples of
hydrophilic ointment bases are solid macrogols (polyethylene
glycols). Other examples of ointment bases are triethanolamine
soaps, sulphated fatty alcohol and polysorbates.
[0079] Examples of other excipients are polymers such as carmelose,
sodium carmelose, hydroxypropylmethylcellulose, hydroxyethylcel
lulose, hydroxypropylcellulose, pectin, xanthan gum, locust bean
gum, acacia gum, gelatin, carbomer, emulsifiers like vitamin E,
glyceryl stearates, cetanyl glucoside, collagen, carrageenan,
hyaluronates and alginates and chitosans.
[0080] It is normally preferred that a local effect is obtained for
the polyclonal antibody, Clearance and thereby activity can be
substantially controlled and prolonged by pharmaceutical
compositions such as microspheres, liposomes, complexes of
positively or negatively charged excipients with antibody molecules
of opposite charge.
[0081] Therapeutic Uses of Polyclonal Antibodies
[0082] In a preferred embodiment, the symphobody included in the
present composition is one that reacts with/binds to an inhalant
allergen including conjunctival and nasopharyngeal allergens, as
well as allergens entering the respiratory tract, or otherwise
enters into the body. The preventive or therapeutic inhalation of
polyclonal antibodies, e.g. symphobodies, directed against common
inhalant allergens is aimed directly at eliminating the cause of
the allergy by aiding the blocking, neutralization, and clearance
from the respiratory tract of the allergic causative agent before
allergic sensitization ensues.
[0083] Thus, the present embodiment of the invention concerns the
possibility of neutralizing the effect of allergen inhalation via
polyclonal antibody inhalations by blocking allergen epitopes
otherwise available for the binding of IgE molecules. Also, the
binding of polyclonal antibodies is predicted to exert a clearance
effect on allergens by mediating the phagocytosis and degradation
of allergens without the induction of allergic responses, as well
as facilitating the upwards clearance away from the respiratory
tract into the pharynx of allergen entrapped in immune complexes
with IgA or IgG together with mucosal mucous, and subsequent
swallowing into the digestive tract.
[0084] Finally, the mucosal administration of allergen-specific
polyclonal antibody, e.g. symphobody of the IgG or IgA isotype,
which are blocking with respect to the binding of allergen-specific
IgE, is hypothesized to inhibit the IgE-mediated antigen
presentation for T lymphocytes which may induce the predominantly
T.sub.H2 type T lymphocyte response to allergens which in allergic
individuals is believed to perpetuate the allergy. Instead, the
presence of blocking allergen-specific polyclonal antibodies, e.g.
symphobodies may result in IgG- or IgA-mediated antigen
presentation for T cells, which in turn may preferentially promote
a T.sub.H1 type T lymphocyte response to allergens, thus
interrupting the vicious cycle of the allergic inflammatory
reaction.
[0085] Allergen epitopes e.g. from pollen are derived from several
proteins, and thus for a single inhalant antibody to be able to
work, it will be required to contain several if not many individual
idiotypic specificities/antigen reactivities. In this respect,
polyclonal antibodies seem far superior to monoclonal
antibodies.
[0086] Consequently, polyclonal antibody compositions may be used
for the prophylaxis or treatment of all types of allergy, including
allergic rhinitis, hay fever, allergic conjunctivitis, and allergic
(extrinsic) asthma, as well as food allergy. In particular, but not
limited to, the polyclonal antibody of the present invention is one
that reacts with/binds to an allergen from: The house dust mites
(e.g. Dermatophagoides farinae or D. pteronyssimus); danders from
cat, dog, or horse; tree pollens from birch (Betula alba), alder,
hazel, oak, willow, plane, beech, elm, maple, ash, and hornbeam;
grass pollens from timothy grass (Phleum pretense), bluegrass (Poa
pratense), rye grass (Lolium perenne), Orchard grass (Dactylis
glomerata), ragweed (e.g. Ambrosia artemisiifolia), sweet vernal
grass (anthoxanthum odoratum), and rye (Secale cereale); or fungi
(e.g. Alternaria, Aspergillus, Cladosporium, and Penicillium). In
addition, allergen-specific polyclonal antibodies, e.g.
symphobodies may be used to treat allergies against other agents
such as food allergens (e.g. peanuts and other nuts, shell-fish,
egg, milk, corn) or bee venom allergens. Many of these allergens
may be purchased as well-characterized proteins from commercial
suppliers.
[0087] The dose of polyclonal antibody required in humans to be
effective in the treatment or prevention of allergy differs with
the type and severity of the allergic condition to be treated, the
type of allergen, the age and condition of the patient, etc.
Typical doses of polyclonal antibody to be administered are in the
range of 1 .mu.g to 1 g, preferably 1-1000 .mu.g, more preferably
2-500, even more preferably 5-50, most preferably 10-20 .mu.g per
unit dosage form.
[0088] Experimental
[0089] The present invention is described in detail in the
following examples which are not in any way intended to limit the
scope of the invention as claimed.
[0090] Immunization of Mice for the Generation of Symphobody
Libraries
[0091] BALB/c mice are immunized subcutaneously (s.c.) or
intraperitoneally (i.p.) with e.g. 1 mg of allergenic protein in
Freunds complete adjuvant. Immunization is performed using
recombinant allergen protein (e.g. Der p 1) or extracts from native
allergens. Any subsequent immunizations are given at two to three
week intervals and in incomplete Freunds adjuvant. Spleen and/or
bone marrow are taken 3 days after the last immunization and used
for the preparation of the symphobody library, as described in U.S.
Pat. No. 5,789,208.
[0092] Generation of Symphobody Libraries from Allergic Patient
Material
[0093] Symphobody libraries are prepared from blood or bone marrow
samples taken from allergic patients characterized by positive case
history, skin prick testing, radioallergosorbent test (RAST), or
reactivity of patient sera with allergen extracts by IgG or IgE
immunoblotting or reactivity to purified recombinant allergens
(e.g. pollen allergens or animal allergens).
[0094] Antibody Binding to Allergen is Detected By ELISA
[0095] Between 50 and 1000 ng of allergen, disintegrated allergen,
or recombinant allergen are coated pr well of Nunc Maxisorp 96-well
microtiter plates, After washes in PBS containing gelatin or BSA as
well as Tween-20 the wells are blocked 1 hour at 37.degree. C.
using gelatin or BSA. Subsequently the wells are washed and
incubated with either polyclonal antibodies, e.g. symphobodies,
murine or human IgE, IgG derived from either serum or
bronchoalveolar lavages (BAL). After repeated rounds of washing,
bound antibody is detected by successive incubations of secondary
biotinylated anti-mouse or anti-human immunoglobulin as
appropriate, followed by AP-avidin, and pNPP substrate. Previously
characterized allergen-specific monoclonal antibodies are used as a
positive control and monoclonal and polyclonal antibodies with
different, unrelated specificities are used as negative
controls.
[0096] In some experiments polyclonal antibody incubations are
preceded by incubations with well-characterized monoclonal
antibodies in a competitive ELISA.
[0097] Polyclonal Antibody Inhibition of Binding of Patient-Derived
IgE to Allergens
[0098] Patient-derived IgE binding to allergen extracts is studied
either in competitive ELISA (similar to the protocol above with the
following modifications) for IgE binding or by preparative SDS-PAGE
and Western blotting. After ELISA well coating or allergen
electrophoresis using allergen, disintegrated allergen, or
recombinant allergen, the allergen-coated surface is blocked with
gelatin or BSA, before incubation 3-4 hours at 4.degree. C. with
allergen-specific polyclonal antibodies. Subsequently, samples are
incubated 3-4 hours at 4.degree. C. with patient sera or BAL IgE
diluted 1:5 and bound human IgE antibodies are detected with e.g.
.sup.125I-labeled anti-human IgE antibodies (RAST; Pharmacia) and
visualized by autoradiography. Binding of mouse IgG is detected as
described above.
[0099] Characterization of Polyclonal Antibody Reactivity with
Allergen Extracts By Electrophoresis and Western Blotting
[0100] Allergen extracts are separated by SDS-PAGE and
immunoblotted onto nitrocellulose strips before incubation with the
antibody preparation (patient sera, mouse sera, polyclonal
antibodies, e.g. symphobodies, or control monoclonal antibodies).
In some experiments, the cross-reactivity of polyclonal antibodies
generated against one allergen is examined by testing in ELISA or
Western blotting against a panel of homologous allergens.
[0101] Inhibition of Allergen-Induced Histamine Release from Human
Basophile Granulocytes After Preincubation of Allergens with
Polyclonal Antibodies
[0102] Heparinized blood samples are obtained from allergic
patients and granulocytes isolated by dextran sedimentation.
Recombinant allergens, disintegrated allergens or allergen extracts
are preincubated with allergen-specific polyclonal antibodies, e.g.
symphobodies, or control antibodies or buffer alone, for 1 h at
room temperature before incubation at different concentrations (1,
0.1, 0.01, and 0.001 .mu.g/ml) with granulocytes disintegrated in
histamine release buffer (20 mM PIPES, pH 7.4, 110 mM NaCl, 5 mM
KCl, 1 mM CaCl.sub.2, 1 g/L glucose, 0.3 mg/ml human serum
albumin). Histamine release into the cell-free supernatant is
determined by radioimmunoassay and expressed as a percentage of
total histamine release after cell lysis.
[0103] Polyclonal Antibody Inhibition of Allergic Inflammation in a
Mouse Model of Allergy
[0104] Mice (e.g. BALB/c mice, are sensitized to allergens (e.g.
ragweed allergen) by two or more i.p. injections of allergen (e.g.
150 microgram) and alum on e.g. days 0 and 4. On e.g. day 11 and in
a two to four week timespan, an intratracheal or intranasal
allergen challenge is performed on anesthetized mice where after
mice are analyzed as described below. In some experiments a mouse
model based on ovalbumin (OVA)-sensitization is employed. Briefly,
BALB/c mice are injected i.p. with e.g. 5-100 .mu.g OVA (chicken
egg albumin grade V,Sigma) in 2 mg aluminum hydroxide adjuvant
(alum, Pierce) on day 1 and day 14, before challenge on protocol
days 28, 29 and 30 with either 1% aerosolized OVA in PBS for 20
minutes using a ultrasonic nebulizer (DeVilbiss Somerset, Pa, USA)
or 5-100 .mu.g OVA in 40 .mu.L PBS injected intratracheally in
anesthetized mice. Control mice receive the same amount of PBS.
[0105] On day 32, 24 hours after antigen challenge, 12 mice are
subjected to an airway responsiveness test and killed on day 33. In
the control group 8 mice are used.
[0106] The left lung is tied of and BAL of the right lung is
obtained by 5 repeated washings with 200 .mu.L PBS. The left lung
is fixed and embedded in paraffin for lung histology. A blood
sample (tail blood) is also taken from each mouse and stored at
-80.degree. C. until analysis is carried out.
[0107] In experiments where the ability of allergen-specific
polyclonal antibodies, e.g. symphobodies to inhibit allergic
inflammation is examined, the allergen-specific polyclonal antibody
preparationin doses varying from 1 .mu.g to 1 mg is administered
before, during, or after the administration of the challenge dose
of antigen.
[0108] Polyclonal antibodies with different or unrelated
specificities as well as PBS is used as a negative control, and the
effect is in some experiments compared with a positive control
allergen-specific monoclonal antibody.
[0109] Efficacy Evaluation of Polyclonal Antibodies in Blocking the
Allergic Response in the Murine Allergy Model
[0110] Upon completion of the allergen challenge, the allergic
reaction is evaluated by performing bronchial lavage (BAL) on
euthanized mice, and the BAL fluid is examined by differential
counting for the content of eosinophils, neutrophils, lymphocytes,
and macrophages.
[0111] The lower and upper lobes of the left lung are collected and
fixed in Carnoy's solution (6.times. ethanol; 3.times. acetic acid
glacial; 1.times. chloroform) at 20.degree. C. for .about.15 hours.
After embedding in paraffin the tissues are cut into 4-5 .mu.m
sections. From each mouse 10 airway sections randomly distributed
are assessed for severity of the cellular inflammation and mucus
occlusion. The cellular infiltrate of the peribronchial and
perivascular areas is evaluated semi-quantitatively for the
presence of leukocytes (eosinophils, iymphocytes), quantified on a
scale from 0-5 with an increment of 0.5. Mucus occlusion of the
bronchial lumen is assigned a score using the following measures 0,
0-10% occlusion; 1, 10-30% occlusion; 2, 30-60% occlusion; 3,
60-90% occlusion; 4, 90-100% occlusion. Damage to the airway
epithelium is also estimated on an equivalent scale. All
evaluations are performed by individuals blinded to the protocol
design and the results are recorded photographically. The tissue
sections are stained with hematoxylin and eosin for cellular
staining or hematoxylin and periodic acid-Schiff for mucus
staining.
[0112] Total and OVA-specific IgE, IgG, IgG.sub.1, IgG.sub.2a and
IgG.sub.3 levels in the blood of mice are determined by ELISA as
described above.
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