Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
2001-12-04
2003-12-09
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
C435S219000, C530S370000, C530S372000, C530S379000, C530S412000, C530S416000, C530S417000, C530S427000
Reexamination Certificate
active
06660495
ABSTRACT:
SUMMARY OF THE INVENTION
The present invention relates to a process for the controlled enzymatic cleavage of purified and depigmented active allergenic proteins from indoor and outdoor source materials, which process produces fragments of allergens that retain the natural T-lymphocyte stimulating epitopes, but are depleted of IgE-binding B-cell epitopes and complement-activating agents. The invention also relates to the new pharmaceutical products. These allergen fragments do not exhibit the disadvantages of conventional allergenic extracts for immunotherapy and can be safely used to induce a state of specific T-cell anergy and immunological tolerance in allergic human beings.
GENERAL BACKGROUND
Aqueous extracts of various environmental substrates, like house dust, the shed epithelial debris of animal skins, the pollen grains of grasses, weeds, trees, and several other materials are widely used for the in vivo and in vitro diagnosis of allergic diseases like bronchial asthma, vasomotor rhinitis and pollinosis in predisposed human, so-called “atopic” patients. Since the first clinical reports by Noon and Freeman in 1911, such extracts have also been applied for the treatment of atopic allergic diseases in a regimen of subcutaneous injections for the “desensitization”, “hyposensitization”, or “immunotherapy” of these ailments. Based on the observation that the causative and predominant allergenic components in the extracts comprise protein backbone structures in the molecular weight range of 10-70 kDa, it has become common practice in the manufacturing process of allergenic extracts to dialyse or ultrafilter the aqueous extracts through membranes of 10 kDa nominal cut-off in order to remove less relevant components with a molecular size lower than 10 kDa, thereby retaining the multivalent antigenic proteins in the range of 10-100 kDa in order to improve the quality of the allergenic extracts for clinical application. In some cases, a lower cut-off limit is chosen of 3 kDa or 5 kDa.
According to current theory the exposure to environmental allergens in man causes the formation of allergen-specific antibodies of the IgG- and IgE-isotypes. Within an immunological context, allergens may therefore be regarded as ordinary IgG-antibody inducing foreign antigens endowed with the ancillary property—either due to special molecular characteristics or to the simultaneous presence in allergenic extracts of appropriate modulators—to additionally promote the biosynthesis of antibodies of the IgE class. At one time, such antigens were therefore more appropriately called “atopic allergens” [1]. Genetically predisposed, so-called “atopic” human beings are particularly prone in this respect to respond with elevated levels of allergen-specific IgE antibodies. After the initial induction or “sensitization” phase, renewed contact with the allergen is considered to lead to the local formation of allergen—(IgE) antibody complexes being responsible for the ultimate disease symptoms. More particularly, it is the interaction of allergens with their specific IgE-class sessile antibodies bound to IgE-(e) receptors on cell membranes (i.e. of mast cells or basophilic leucocytes) which is thought to trigger the sequence of biochemical events eventually leading to the release of mediator substances responsible for the clinically manifest symptoms of allergy. In order to reduce the risk of anaphylactic side-reactions during immunotherapy a large number of approaches has been described. Recently, PCT/EP96/01733 was published (Nov. 7, 1996) disclosing a novel pharmaceutical composition for use in desensitization therapy of allergy sufferers. This composition comprises tyrosine and a polymerised allergen. Other approaches were presented already in the early eighties but were found inadequate. GB-A-1,492,973 describes a coprecipitate of tyrosine and an allergen that has been modified by an agent causing intramolecular crosslinking. EP-A-0.367.306 describes a process of preparing polymerised allergens also providing crosslinking and purportedly resulting in reduced allergenicity. In yet another old publication EP-B-0.038.154 which was published already in 1981 the treatment of an allergen with polysarcosine to reduce its allergenicity is described. In particular polysarcosine with a molecular weight of 2,000-12,000 is useful according to the publication. This document also refers to UK patent 1.282.163 describing an improved form of therapy wherein the allergenicity of an allergen is reduced by treatment with glutaraldehyde, which is suggested as retaining the ability to elicit blocking antibodies but exhibiting reduced allergenicity, thus rendering it more suitable for desensitization therapy. An alternative approach was provided in UK patent no 1.578.348 wherein it is stated that allergen-polyethylene glycol conjugates are capable of eliciting the desired therapeutic effect of suppressing allergen specific IgE production. These materials are stated as being non allergenic and non immunogenic. For the intended immunotherapy of allergic diseases by means of tolerogenic allergen fragments, the products of choice should be closely similar to the natural T-cell epitopes generated within so-called “antigen-presenting cells” (APCs) by acid hydrolases and other enzymes under physiological conditions. This particular approach has been taken in the prior art in U.S. Pat. No. 4,469,667 and European Patent EP 0113712 B1 (see also ref. 6 and 7). Such natural elements include possible peptide-bound haptenic chemical structures and may be imitated in the laboratory by subjecting the allergenic extract components to proteolytic cleavage under controlled conditions. One of the many routes of research in this field suggests that the treatment of allergy in man should be performed using allergen derivatives which do not induce specific IgE antibodies, i.e. allergen derivatives depleted of their so-called IgE-binding “B-cell epitopes”.
Thus, a large number of different ways to provide compositions for desensitization, i.e. by reduction of allergenicity, has been suggested over the past twenty years. Considerable effort has gone into addressing the problem in various ways, so far with little result. In order to get a better understanding of the requirements for treatment, it is essential to assess what actually happens during the induction or elicitation of an allergic response.
Induction of an immune response to foreign antigens requires the cooperation of various categories of cells, i.e. in the simplest form the so-called “antigen-presenting cells” (APC) and the T- and B-lymphocyte populations. The first signal, namely between APCs and T-lymphocytes, is composed of antigenic peptide fragments which arise during the intracellular processing (by proteolytic cleavage) of a foreign antigen internalized by APCs, e.g. macrophages. The (foreign) antigenic fragments (also known as “T-cell epitopes”) are subsequently recycled to the outer membranes of the APC where they then become exposed in the form of ligands associated with membrane-bound Class II antigens of the Major Histocompatibility Complex (MHC). From among the populations of T-(helper) lymphocytes (Th1-, and Th2-cells) recognizing such T-cell epitopes on APCs and cooperating by themselves with immunoglobulin-secreting B-lymphocytes, activation of the allergen-specific Th2-cell clones in particular is considered to exercise a pivotal role in the development of human atopic diseases. Unlike Th1-cells, Th2-cells activated by contact with APCs expressing the proper T-cell epitopes on their surface produce relatively large amounts of the interleukins 4 (IL-4) and 5 (IL-5), which may among others act as cytokine signals promoting IgE biosynthesis by B-lymphocytes or plasma cells. Based on this, a contemporary theory of “immediate Type I” (atopic) allergy purports that in atopic human beings a relative preponderance of Th2 over Th1 lymphocytes occurs, resulting in increased IgE synthesis, and that active therapy should therefore aim at restoring the balance of allergen-specifi
Berrens Lubertus
Gallego Camara Maria Teresa
Gonzales Romano Maria Leticia
Browdy and Neimark , P.L.L.C.
C.B.F. Leti S.A.
Lilling Herbert J.
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