Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Hormone or other secreted growth regulatory factor,...
Reexamination Certificate
1998-12-04
2002-09-24
Eyler, Yvonne (Department: 1646)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Hormone or other secreted growth regulatory factor,...
C424S178100, C530S300000, C530S328000, C530S350000, C530S399000, C530S388220, C536S023500, C514S002600
Reexamination Certificate
active
06455049
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an active biological molecule of the peptide type which when it is linked covalently to certain transporter peptides and/or certain adjuvants is capable of inducing in vivo a potentiating effect on the biological activity of growth hormone GH.
2. Description of the Related Art
The potentiation of the biological activity, more particularly of the somatogenic activity, of GH by antibodies was first observed by Holder et al. (1980), who based their studies on the measurement of incorporation of radioactive sulfates into costal cartilage as well as on the weight gain induced by GH in Snell type dwarf mice.
Three studies based on the same parameters performed by Holder et al. (1985) and Aston et al. (1986 and 1987) showed that the majority of the antibodies studied had a potentiating effect on the biological action of GH. The work of Wallis et al. (1987) confirmed the phenomenon of enhancement of the somatogenic activity of bovine growth hormone by anti-GH antibodies. These latter based their studies on the measurement of the IGF-1 (insulin growth factor) plasma level and the weight gain of hypophysectomized rats of the Wistar strain.
In particular, the European patent application EP 284 406 (COOPERS ANIMAL HEALTH LTD.) described a peptide having a primary structure homology with a continuous sequence of amino acid residues of the growth hormone in the region extending from positions 35 to 53 or peptides with cross-reactivity, said peptide being usable in an antigenic composition to potentiate the effects of growth hormone in a vertebrate. The experiments described by Ashton et al. consist of an in vivo administration of antibodies, either in the form of sera when the latter are obtained from sheep, or in the form of monoclonal antibodies and this passive administration of antibodies directed against the peptide or directed against native natural growth hormone, complexed with said hormone, increases the biological activity of the latter. It has not been demonstrated that the peptide 35-53 of growth hormone could be used in the framework of active immunization.
Recent studies performed with potentiating antibodies would seem to indicate that if GH alone binds to hepatocytes, the GH-MAb complex or the complex formed between the hormone according to the invention and a monoclonal potentiating antibody would bind preferentially to the sinusoidal cells (Kupffer cells) (Tans et al. (1994)). Thus, it might consequently be assumed that the synthesis of IGF-1 and IGF BP3 in the hepatocytes (Massart et al. (1993)) might also be enhanced by the action of said complexes at these sinusoidal cells.
It is described in the patent application EP 137 234 (THE WELLCOME FOUNDATION LIMITED) that certain antibodies to growth hormone are capable of potentiating the activity of the latter whereas it is known that, usually, such antibodies have a tendency to antagonize its action, at least in vivo. Such antibodies may in addition be produced in situ by the “vaccination” of the host animal with a specific fragment of growth hormone, such that a class of polyclonal antibodies of restricted specificity is created which might potentiate the activity of the endogenous hormone.
BRIEF SUMMARY OF THE INVENTION
In the present invention it is shown that certain sequences of growth hormone linked to a carrier molecule and/or an adjuvant are capable of potentiating the activity of said hormone in a vertebrate. The discovery of the peptide fragment of the present invention is the result of research based initially on the selection of monoclonal antibodies (MAb) of the IgG class, whose reactivities towards growth hormone, in particular bovine growth hormone, appeared to be the highest. After purification of said MAb, four types were selected. The effect of these latter on the plasma IGF-1 response induced by a single injection of bGH into the immature hypophysectomized rat was assayed by Massart et al. (1993). Finally, it was possible to identify an epitope recognized by a potentiating antibody designated 2H4, this antibody being capable of specifically recognizing a peptide sequence situated in the &agr;-helix No. 3 of native growth hormone.
Every reference to the primary, secondary or tertiary structure of GH refers to that described in Scanes C.G. et al. (1995).
The objective of the present invention is to provide a means for potentiating growth hormone GH more effectively and more durably than those of the prior art. In particular, the present invention provides a peptide or hapten capable of inducing in vivo a potentiating effect of the biological activity of this hormone when it is covalently linked to a transporter peptide and/or an adjuvant.
Such an effect may be the result of active immunization of the vaccination type, in which the administration of the hapten induces, in particular, the production of specific antibodies in vivo.
DETAILED DESCRIPTION OF THE INVENTION
The present invention thus relates to a peptide construction comprising all or part of the sequence extending from position 103 to position 114 of the growth hormone GH or of a homologous peptide of said sequence provided that it exhibits an immunological cross-reactivity with said sequence, this peptide fragment being linked covalently to a transporter peptide and/or an adjuvant and being capable of inducing in vivo a potentiating effect on the biological activity of said growth hormone.
The precise identification of this GH sequence or “GH peptide” resulted from the analysis of a certain number of anti-GH monoclonal antibodies, responsible for a potentiating effect on the administration of the antibody-GH complex. This peptide fragment, the peptide sequence of which derived from GH may also be considered as a hapten-type molecule, is recognized by the antibody 2H4. Moreover, among the antibodies having the highest potentiating effects it may be noted that the affinity of 2H4 for GH is low compared with those of other antibodies.
According to a preferred peptide construction of the invention, said sequence of growth hormone GH is comprised in the following sequence:
GTSDRVYEKL SEQ ID NO:1.
Even more preferably, said sequence of the GH is selected from the following sequences:
TSDRVYEKL SEQ ID NO:2;
GTSDRVYEK SEQ ID NO:3;
SDRVYEKL SEQ ID NO:4;
TSDRVYEK SEQ ID NO:5; or
GTSDRVYE SEQ ID NO:6.
These five peptides correspond to the regions 105-113, 10-112, 106-113, 105-112, 104-111, respectively, of the growth hormone GH as published in Scanes et al. (1995) and may be obtained by the standard procedures of peptide synthesis.
Since a hapten is, by definition, non-immunogenic, the peptide of the invention is coupled covalently to a transporter. As examples, such transporters may advantageously be peptide sequences derived from ovalbumin, KLH (keyhole limpet hemocyanin) or albumin and more particularly the following peptides:
323-339 of ovalbumin
378-398 or 379-398 or 378-397 or 378-396 or 378-395 of the peptide CS.T3 described by Sinigaglia et al.,
45-60 of protein 1A of the respiratory syncitial virus
120-140 of the protein enveloping the genomic RNA of the hepatitis B virus.
These sequences are coupled covalently to the carboxyl or amino terminal of the “GH peptide”.
The peptide fragment of the invention may be linked covalently to an adjuvant of the N-acetyl-muramyl (MAP) type, linked to a peptide like MDP or its derivatives.
According to a preferred embodiment of the peptide fragment according to the invention, the adjuvant is the muramyl-dipeptide (MDP) or one of its derivatives such as MDP-lysine, Lys (NH
2
)—D—isoGlu—L—Ala—NMc—Nur.
The MDP derivative is then coupled covalently to the carboxyl end of the peptide consisting of the “GH peptide” and the transporter, approximately similar to that described in EP 89290.
The peptide fragment according to the invention may also be linked to a sequence known or presumed to be T-dependent with or without MDP or its derivatives or alternatively linked or incorporated into liposomes. It may also be administered in aqueous
Portetelle Daniel
Renaville Robert
Bieker-Brady Kristina
Brannock Michael T
Clark & Elbing LLP
Eyler Yvonne
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