Synthetic peptides derived from the beta-subunit of human thyroi

Details Synthetic peptides derived from the beta-subunit of human thyroi Synthetic peptides derived from the beta-subunit of human thyroi

1991-07-25

1993-03-23

Draper, Garnette D.

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

11 to 14 amino acid residues in defined sequence

530397, 530326, 530398, C07K 700

Patent

active

051965135

DESCRIPTION:

BRIEF SUMMARY
55, .beta.71-85, .beta.81-95 and .beta.101-112. These polypeptides inhibit binding of TSH to human thyroid membrane.
The most preferred polypeptides are: (I) Phe-Cys-Ile-Pro-Thr-Glu-Tyr-Thr-Met-His-Ile-Glu-Arg-Arg-Glu; (II) Leu-Ser-Cys-Lys-Cys-Gly-Lys-Cys-Asn-Thr-Asp-Tyr-Ser-Asp-Cys; and (III) Lys-Thr-Asn-Tyr-Cys-Thr-Lys-Pro-Gln-Lys-Ser-Tyr corresponding respectively to isolated .beta.-subunit residues 1-15, 81-95, and 101-112 of human TSH. In the case of (I) and (II), C-terminal amides of these polypeptides are most preferred. These polypeptides were assayed for bioactivity and found to effectively inhibit TSH-mediated cAMP generation, and demonstrate an inhibitory effect on thyroid stimulating immunoglobulin obtained from sera of patients with Graves' disease to stimulate cAMP production by FRTL.sub.5 cells.
Since it is expected that further digestion/hydrolysis of polypeptides of the present invention in vitro or in vivo will yield fragments of substantially equivalent bioactivity, such lower molecular weight polypeptides are considered to be within the scope of the present invention, as are mixtures of the polypeptides and functional analogues of the polypeptides.
The polypeptides of the present invention also possess characteristics which can be applied for immunodiagnostic and immunotherapy purposes including competitive and non-competitive assays for thyroid stimulating immunoglobulin. Antibodies raised in response to polypeptides of the present invention can be used as an immunodiagnostic to measure TSab in a patient sample. Also, antibodies raised in response to polypeptides of the present invention can be used therapeutically to neutralize TSab.


BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the strategy used to make synthetic overlapping .beta.TSH peptides. Exact amino acid sequences are shown in Table 1.
FIG. 2 is a graph depicting inhibition of .sup.125 I-bTSH binding to human thyroid membrane by synthetic .beta.-subunit peptides (human thyroid membrane TSH radio-receptor assay).
FIG. 3 is a graph depicting inhibition of .sup.125 I-bTSH binding to porcine thyroid membrane by synthetic .beta.-subunit peptides (porcine thyroid membrane TSH radio-receptor assay).
FIG. 4 is a graph depicting lack of inhibition of .sup.125 I-bTSH binding to porcine thyroid membrane by synthetic .beta.-subunit peptides (porcine thyroid membrane epidermal growth factor radio-receptor assay.)
FIG. 5 is a graph depicting the standard curve for TSH stimulated cAMP production in FRTL.sub.5 cells.
FIG. 6 is a graph depicting inhibition of TSH-mediated cAMP generation in FRTL.sub.5 cells by synthetic human TSH .beta.-subunit peptides.
FIG. 7 is a graph depicting inhibition of thyroid stimulating immunoglobulin mediated cAMP generation in FRTL.sub.5 cells by synthetic .beta.-subunit peptides 102-112, 1-15, 81-95 and 91-105. Each line represents a serum sample from a different patient with Graves' disease.
FIG. 8 is a graph depicting inhibition of thyroid stimulating immunoglobulin stimulated-mediated cAMP generation by synthetic .beta.-subunit peptides 71-85, 31-45 and 61-75. FIG. 8 shows peptide 71-85 was effective only at the highest dose of serum tested while peptides 31-45 and 61-75 did not produce a significant effect.


DETAILED DESCRIPTION OF THE INVENTION

We prepared eleven synthetic beta (.beta.)-subunit peptides to study the structure-function relationships of the peptide sequence of the TSH hormone. The receptor binding and biologic activity of these synthetic .beta.-subunit peptides in a human thyroid membrane system and the functional rat thyroid follicular cell line FRTL.sub.5 were evaluated. Specifically, the ability of the synthetic peptide fragments of the .beta.-subunit of TSH to interact with the TSH receptor on human thyroid membranes was studied. Selected fragments were evaluated for ability to inhibit TSH-mediated cAMP generation and blocking action of long-acting thyroid stimulator (known as LATS or TSab, thyroid stimulating immunoglobulin).


Synthesis of Polypeptides

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This invention was made with Government support under grant number HD 9140 by the National Institute of Health and funds from the Mayo Foundation. The Government has certain rights in the invention.
This is a continuation of application Ser. No. 07/403,564, filed Sept. 5, 1989, now abandoned.

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