Immunogenic compositions that potentiate growth hormone...

Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Hormone or other secreted growth regulatory factor,...

Reexamination Certificate

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C514S002600, C514S012200, C530S300000

Reexamination Certificate

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06328974

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to synthesis, cloning, and expression of fused proteins. More specifically, this invention relates to peptides comprising epitopes of porcine somatotropins (pST) fused to a non-related peptide and the use of the resulting fused protein to potentiate pST activity following immunization.
BACKGROUND OF THE INVENTION
In what may be considered to be, at first blush, counterintuitive, it has been appreciated for some time that antisera raised against hormone antigens are not necessarily inhibiting but can, in fact, potentiate hormone activity in vitro and in vivo (Thompson, K. W.,
Proc. Soc. Exp. Biol. Med
. 35:640-44 (1937) and Rowlands, I. W.,
J. Endocrinol
. 1:177-183 (1939). Antibody-mediated enhancement of hormone activity has been reviewed by Aston et al., (
Molecular Immunol
. 26(5):435-446 (1989)), the contents of which are incorporated herein by reference to more fully describe the background of this invention. Of relevance to this invention are the observations regarding the beneficial effect of antibodies raised to growth hormones, more particularly, porcine growth hormone.
The amino acid sequence has been determined for growth hormone from numbers of species including human growth hormone (hGH), porcine (pGH), bovine (bGH), horse (hoGH), rat (rGH), monkey (mGH), avian (aGH), fish (fGH), canine (cGH), and ovine (oGH) (See for example: Seeburg, P. H. et al.,
DNA
2(1):37-45 (1983); Abdel-Mequid, S. S. et al.,
Proc. Nat'l. Acad. Sci
. (USA) 84:6434-37 (1987)). Growth hormone, also referred to in the literature as somatotropin (ST), is a 22,000 dalton protein secreted by the anterior pituitary gland in mammals. In its native form the molecule contains 190 amino acids. Because the native form results from the cleavage of a 26 amino acid signal sequence from a larger precursor molecule, there can be some NH
2
-terminal length polymorphism due to inefficient post-translational processing. Accordingly, both 190 amino acid NH
2
-terminal phenylalanine and 191 amino acid NH
2
-terminal alanine forms are known (Mills, J. B. et al.,
J. Biol. Chem
. 245:3407-15 (1970)). Although some variation occurs, the above-mentioned somatotropins share a good deal of structural and functional homology such that epitope regions identified in one species are good predictors for analogous regions in another species.
The nucleotide and amino acid sequences of pST are reported in Seeburg et al. (supra) including 20 amino acids of the leader peptide. The nucleotide and amino acid sequences represented by Sequences I.D. Nos. 1 and 2 respectively.
Aston et al., reported the potentiation of the somotogenic and lactogenic activity of hGH (
J. Endocrinol
. 110:381-388 (1986) and bGH (
Mol. Immunol
. 24(2):143-150 (1987)) by monoclonal antibodies when given in combination with these hormones.
EP Application 0284406, published Sep. 28, 1988, relates to certain growth hormone fragments spanning positions 35 to 53 and antigenic formulations thereof useful in potentiating the effects of growth hormone.
Similarly, PCT Application WO89/001666, published Jan. 12, 1989, relates to antibodies raised to antigenic peptides spanning the region 112-159 of native growth hormone and portions thereof coupled to a carrier. The antibodies were shown to potentiate growth hormone effects. Porcine growth hormone regions 134-154 and 120-140 and fragments thereof were specifically identified.
EP Application 0303488, published Feb. 15, 1989, specifically identifies the following regions 1 to 18, 55-72, 97-110, 119-131, 122-138, 123-137, 130-143, and 133-146 as useful as components of antigenic formulations. The antibodies generated to the foregoing peptides have growth hormone enhancing effects.
EP Application 0492788, published Jun. 5, 1991, identifies additional pST regions said to be useful for generating antibodies in pigs and rabbits. Regions specifically mentioned include 98-110, 110-118, and 155-163.
More recently, the epitopes on bGH have been identified as being included within regions 120-140 and 134-154. Aston et al.,
Mol. Immunol
. 28(1/2):41-50 (1991).
The individual peptide epitopes, such as those described above, are known to function individually. Furthermore, these fragments were chemically synthesized and as such were not amendable to scale-up for commercial vaccine production. This invention employs a recombinant DNA approach in which at least two distinct epitopes are biosynthesized as a composite peptide molecule. This molecule can be then coupled chemically to a carrier to enhance immunogencity, or more preferrably, the DNA encoding the composite molecule can be operatively linked, in frame, with the DNA encoding a non-related peptide; the entire construction upon expression results in a fusion protein which then may be used directly or be optionally chemically coupled to a carrier.
BRIEF DESCRIPTION OF THE INVENTION
This invention relates to composite peptides comprising at least two non-contiguous somatotropin epitopic amino acid sequences.
This invention also relates to fusion proteins comprised of a composite somatotropin peptide linked to a non-related protein.
This invention also relates to both composite peptides or fusion proteins optionally linked to a carrier molecule.
This invention further relates to isolated DNAs encoding the aforesaid composite peptides and fusion proteins, expression vectors comprising those DNAs and host cells transformed by the expression vectors.
This invention further relates to vaccines comprising the composite peptides or fusion proteins.
This invention also relates to the recombinant production of the composite peptides and fusion proteins.
This invention also relates to a method for potentiating the action of growth hormone in a pig comprising inducing an antibody response to the composite peptide and treating said induced pig with a growth enhancing amount of pST.


REFERENCES:
patent: 4425437 (1984-01-01), Riggs
patent: 4578355 (1986-03-01), Rosenberg
patent: 5210180 (1993-05-01), Wang et al.
patent: 5338836 (1994-08-01), Wang et al.
patent: 5506107 (1996-04-01), Cunningham et al.
patent: 5547669 (1996-08-01), Rogers et al.
Cunningham and Wells, 1989, High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis, Science 244: 1081-1085.
Germino and Bastia, 1984, Rapid purification of a cloned gene product by genetic fusion and site-specific proteolysis, Proc. Natl. Acad. Sci. USA, 81: 4692-4696.
Shen, 1984, Multiple joined genes prevent product degradation inEscherichia coli, Proc. Natl. Acad. Sci. USA, 81: 4627-4631.
Aston et al., 1987, Enhancement of bovine growth hormone activity in vivo by monoclonal antibodies, Mol. Immun., 24: 143-150.
Li et al., 1974, Human pituitary growth hormone: isolation and properties of two biologically active fragments from plasmin digests, Proc. Natl. Acad. Sci. USA 71:1197-1201.
Aston et al., 1983, Antigenic, receptor-binding and mitogenic activity of proteolytic fragments of human growth hormone, EMBO, 2:493-497.
Seeburg et al, 1983, Efficient bacterial expression of bovine and porcine growth hormones, DNA 2:37-45.
Young et al., 1983, Efficient expression of influenza virus NS1 nonstructural proteins in E. coli, Proc. Natl. Acad. Sci. USA 80:6105-6109.
Aston et al., 1991, Antigenic structure of a bovine growth hormone: location of a growth enhancing region, Mol. Immunol. 28:41-50.

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