JP4081130B2 - Glycoprotein hormone super agonist - Google Patents

Description

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates generally to modified glycoprotein hormones. In particular, the present invention relates to modifications to human glycoproteins that produce superagonist activity.

BACKGROUND ART Thyrotropin (thyroid stimulating hormone, TSH) and gonadotropin, chorionic gonadotropin (CG), lutropin (luteinizing hormone, LH), and follitropin (follicle stimulating hormone, FSH) constitute a family of glycoprotein hormones. To do. Each hormone is a heterodimer of two non-covalent subunits, α and β. Within the same species, the amino acid sequence of the α-subunit is identical for all hormones, while the sequence of the β-subunit is hormone specific (Pierce, JG and Parsons, TF. “Glycoprotein hormones: strcture and function. "Ann. Rev. Biochem. 50: 465-495 (1981)). The fact that subunit sequences are highly conserved from fish to mammals means that these hormones have evolved from a common ancestral protein (Fontaine YA. And Burzawa-Gerard, E. “Esquisse de l ' evolution des hormones gonadotopes et threotropes des vertebres. "Gen. Comp. Endocrinol. 32: 341-347 (1977)). Evolutionary changes in these hormones have in some cases resulted in a modification of biological activity (Licht, P. et al., “Evolution of gonadotropin structure and function.” Rec. Progr. Horm. Res., 33: 169-248 (1977) and Combarnous, Y. “Molecular basis of the specificity of binding of glycoprotein hormones to their receptors.” Endocrine Rev. 13: 670-691 (1992)). The determinants are not clear. For example, human thyroid stimulating hormone (hTSH) and bovine thyroid stimulating hormone (bTSH) have a high degree of homology in the α (70%) and β (89%) subunit sequences, but bTSH is 6-10 times greater than hTSH. Effective (Yamazaki, K. et al., “Potentthyrotropic activity of human chorionic gonadotropin variants in terms of ¹²⁵ I incorporatin and de novo synthesized thyroid hormone release in human thyroid follicles.” J. Clin. Endocrinol. Metab. 80: 473 -479 (1995)).

  Glycoprotein hormones are extremely important in certain treatments, such as the treatment of thyroid cancer patients (eg, Meier, CA, et al., “Diagnostic use of Recombinant Human Thyrotropin in Patients with Thyroid Carcinoma (Phase I / II Study). "J. Clin. Endocrinol. Metabol. 78:22 (1994)). The possibility of using human thyroid stimulating hormone (TSH) in the treatment of this disease has been abandoned because of the potential for transmission of Creutzfeldt-Jakob disease. An alternative to the use of human TSH is the use of bovine TSH, but this approach is based on hormonal anaphylactic shock with relatively high likelihood that the hormone has side effects such as nausea, vomiting, local sclerosis, urticaria. Is very limited (Meier, CA, etc.). The lack of biocompatibility of urinary gonadotropins and the limited efficacy of recombinant glycoprotein hormones are reasons for replacing them with more effective recombinant analogs. Accordingly, there is a need for human derived glycoprotein hormones, as well as agonists of these hormones.

For example, administration of an agonist of thyroid stimulating hormone in certain clinical conditions, such as thyroid cancer, enhances the uptake of radioactive iodine into the cancer to treat the disease. Thyroid-stimulating hormone agonists direct higher amounts of radioactive iodine to cancer, thereby providing a more effective treatment. Alternatively, the glycoprotein hormone used to induce ovulation is replaced with a super agonist. This reduces the need for hormones today, a major medical problem in fertility treatment (Ben-Rafael, Z., et al., “Pharmacokinetics of follicle-stimulating hormone: clinical significance.” Fertility and Sterility 63 : 689 (1995)). If the use of wild-type follicle-stimulating hormone results in over-stimulation and high rates of multiple pregnancy and miscarriage due to a large number of hormone molecules that apparently stimulate a large number of follicles, a follicle-stimulating hormone superagonist is administered. Can treat infertility. The use of this modified hormone agonist may reduce the stimulation frequency of a large number of follicles because a small number of hormone molecules can be administered to achieve the desired result.

  The present invention provides for the first time specific amino acid substitutions of human glycoprotein hormones that result in human glycoprotein hormone analogs that exhibit large increases in both in vitro and in vivo biological activity.

SUMMARY OF THE INVENTION For the purposes of the present invention as embodied and broadly described herein, the present invention, in one aspect, comprises the group consisting of positions 11, 13, 14, 16, 17, and 20. Human glycoprotein hormones having at least three basic amino acids in the α-subunit at selected positions are provided.

  The present invention further provides a human glycoprotein hormone having at least one basic amino acid in the α-subunit at a position selected from the group consisting of positions 11, 13, 14, 16, 17, and 20.

  In another aspect, the present invention relates to a modified human glycoprotein hormone having an activity greater than that of a wild-type human glycoprotein, wherein the non-human glycoprotein hormone has an activity greater than that of the wild-type human glycoprotein. Provided are modified human hormones having the same amino acid substituted at the corresponding position.

  In another aspect, the present invention provides a method of treating a condition associated with glycoprotein hormone activity in a subject, comprising administering to a patient a therapeutic amount of a glycoprotein hormone of the present invention.

  In another aspect, the invention relates to a method of constructing a superactive non-chimeric analog of a human hormone, wherein the amino acid sequence of a more active homolog from another species is compared with the human hormone and Are substituted with corresponding amino acids from other species, the activity of the substituted human hormone is measured, and a superactive analog is selected from the substituted human hormone.

  In yet another aspect, the present invention provides a nucleic acid encoding a modified glycoprotein hormone.

DETAILED DESCRIPTION OF THE INVENTION The present invention is further facilitated by reference to the following detailed description of preferred embodiments of the invention and the examples contained therein, and to the drawings and the foregoing and following description thereof. To be understood.

  Prior to disclosing and describing the compounds, compositions and methods of the present invention, the present invention describes specific hormones, specific subjects, ie humans and non-human mammals, specific amino acids, specific clinical conditions, specific analogs, Or, it should be understood that the invention is not limited to a particular method and can be modified as a matter of course, and numerous modifications and changes will be apparent to those skilled in the art. Furthermore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

  In this specification and in the claims, a word means one or more of it, depending on the context in which it is used. Thus, for example, taking “human glycoprotein hormone” as an example, this means that at least one human glycoprotein hormone is used.

  In one aspect, the invention provides a human glycoprotein hormone having at least three basic amino acids in the α-subunit at a position selected from the group consisting of positions 11, 13, 14, 16, 17 and 20. .

  The present invention further provides a human glycoprotein hormone having at least one basic amino acid in the α-subunit at a position selected from the group consisting of positions 11, 13, 14, 16, 17, and 20.

  In another aspect, the invention corresponds to a basic human amino acid position of a modified human glycoprotein hormone having activity greater than wild type human glycoprotein and having activity greater than wild type human glycoprotein. A modified human hormone having the same amino acid substituted at the position is provided.

  In another aspect, the present invention provides a method of treating a condition associated with glycoprotein hormone activity in a subject, comprising administering to a patient a therapeutic amount of a glycoprotein hormone of the present invention.

  In another aspect, the present invention provides a method of assisting reproduction in a subject comprising administering a supplemental amount of a glycoprotein hormone of the present invention.

  In another aspect, the invention relates to a method of constructing a superactive non-chimeric analog of a human hormone, wherein the amino acid sequence of a more active homolog from another species is compared to the human hormone and the amino acid in the human hormone is Methods are provided that include substituting corresponding amino acids from other species, measuring the activity of the substituted human hormone, and selecting a superactive analog from the substituted human hormone.

A “human” glycoprotein hormone is one in which the number of amino acid substitutions made in the wild-type sequence is the number of amino acid differences at the corresponding position in the corresponding polypeptide hormone between human and another species. / 2 means not exceeding. Thus, based on the amino acid coding sequence, the modified polypeptide hormone is considered more similar to the human wild-type polypeptide hormone than the corresponding polypeptide hormone from a non-human species from which the amino acid substitution is obtained. For example, if there are a total of 20 amino acid differences in the corresponding position of the corresponding glycoprotein hormone between the human glycoprotein and the bovine glycoprotein hormone, then the “human” glycoprotein hormone will have the bovine glycoprotein hormone within its amino acid sequence. A modified wild-type human hormone containing 10 or fewer amino acid substitutions that are homologous to the corresponding amino acid in the amino acid sequence. More specifically, thyroid stimulating hormone is 20 or more of a total of 40 amino acid differences between the α- and β-subunits of human and bovine homologues, as described in the Examples included herein. If the above is homologous to the corresponding amino acid in human thyroid stimulating hormone, it is considered “human”.

  Due to the risk of a bad immune response to administration of the modified glycoprotein hormone when the recipient of the modified glycoprotein hormone is human, the modified glycoprotein hormone is preferably accompanied by an unacceptable loss of superagonist activity. It has homology to the human amino acid sequence to the maximum extent possible. Alternatively, if the subject to whom the modified glycoprotein is administered is non-human, the modified glycoprotein hormone is preferably a specific non-human amino acid sequence to the maximum extent possible without unacceptable loss of superagonist activity. And homology. Therefore, in a preferred embodiment of the present invention, when a wild-type glycoprotein is modified by substituting a specific amino acid to construct a modified glycoprotein having super agonist activity, the substituted amino acid that does not increase agonist activity is: The number is 10 or less, in particular 9, 8, 7, 6, 5, 4, 3 and 2 or 0.

  Similarly, “non-chimeric” means that the number of amino acid substituents does not exceed one half of the number of amino acid differences at the corresponding position of the corresponding polypeptide hormone between species, and thus the modified polypeptide hormone is Based on the amino acid coding sequence, it is believed that it resembles a wild-type polypeptide hormone of a species that is modified from the corresponding polypeptide hormone from the species from which the amino acid substitution is obtained.

  In yet another aspect, the present invention provides a nucleic acid encoding a modified glycoprotein hormone.

  Glycoprotein hormones are a family of hormones that are structurally related heterodimers consisting of an α-subunit common to species and a different β-subunit in species that imparts biological specificity to each hormone. Includes. For a general review of glycoprotein hormones, see Pierce, J.G. et al., “Glycoprotein hormones: structure and function.” Ann. Rev. Biochem. 50: 465-495 (1981) and Combarnous, Y.M. "Molecular basis of the specificity of binding of glycoprotein hormones to their receptors." Endocrine Rev. 13: 670-691 (1992). This family of hormones includes chorionic gonadotropin (CG), lutropin (luteinizing hormone, LH), follitropin (follicle stimulating hormone, FSH) and thyrotropin (thyroid stimulating hormone, TSH). Each of these glycoprotein hormones having at least one basic amino acid in the α-subunit at a position selected from the group consisting of positions 11, 13, 14, 16, 17 and 20 is provided by the present invention.

  Basic amino acids include the amino acids lysine, arginine and histidine, and any other basic amino acids that are modifications to any of these three amino acids, synthetic basic amino acids not normally found in nature, or neutral pH Including any other amino acid that is positively charged.

The glycoprotein hormone provided by the present invention can be obtained in a number of ways. For example, a DNA molecule encoding a glycoprotein hormone can be isolated from a commonly found organism. For example, a genomic DNA or cDNA library is constructed and screened for the presence of the nucleic acid. Such library construction and screening methods are well known in the art, and kits for performing the construction and screening steps are commercially available (eg, Stratagene Cloning Systems, La Jolla, Calif.). Once isolated, the nucleic acid is cloned directly into an appropriate vector or, if necessary, modified to facilitate subsequent cloning steps. Such modification steps are common, and one example is the addition of an oligonucleotide linker containing a restriction site to the nucleic acid terminus. General methods are described in Sambrook et al., “Molecular Cloning, a Laboratory Manual,” Cold Spring Harbor Laboratory Press (1989).

  Once the nucleic acid sequence of the desired glycoprotein hormone is obtained, the basic amino acid can be placed at any specific amino acid position by techniques well known in the art. For example, PCR primers can be designed that span one or more amino acid positions and can replace non-basic amino acids with basic amino acids. The nucleic acid is then amplified and inserted into the wild type glycoprotein hormone coding sequence to obtain any of a number of possible combinations of basic amino acids at every position of the glycoprotein hormone. Alternatively, one skilled in the art can introduce a specific mutation at any point in a specific nucleic acid sequence by techniques for point mutagenesis. General methods are described in Smith, M. et al. “In vitro mutagenesis” Ann. Rev. Gen., 19: 423-462 (1985) and Zoller, M.J. “New molecular biology methods for protein engineering” Curr. Opin. struct. Biol., 1: 605-610 (1991).

Another example of a method for obtaining a DNA molecule encoding a specific glycoprotein hormone is to synthesize a recombinant DNA molecule encoding a glycoprotein hormone. For example, oligonucleotide synthesis methods are common in the art, and oligonucleotides that code for specific protein regions are readily obtained by automated DNA synthesis. Nucleic acids for a single strand of a double-stranded molecule can be synthesized and hybridized with its complementary strand. These oligonucleotides can be designed so that the resulting double-stranded molecule has an internal restriction site or a suitable 5 'or 3' overhang at the end for cloning into a suitable vector. Double-stranded molecules encoding relatively large proteins are easily synthesized by first constructing several different double-stranded molecules encoding specific regions of the protein and then ligating these DNA molecules together can do. For example, Cunningham et al. ("Receptor and Antibody
Epitopes in Human Growth Hormone Identified by Homolog-Scanning Mutagenesis, "Science, 243: 1330-1336 (1989)) first constructed overlapping and complementary synthetic oligonucleotides and ligated these fragments together, A synthetic gene encoding the human growth hormone gene was constructed, Ferretti, et al., Proc. Nat. Acad. Sci. 82: 599-603, which disclosed the synthesis of a 1057 base pair synthetic bovine rhodopsin gene from a synthetic oligonucleotide. See also (1986) By constructing glycoprotein hormones in this way, any particular sugar having a basic amino acid at any particular position in one or more of either the α-subunit, the β-subunit or both. Protein hormones can be readily obtained by those skilled in the art, see also US Pat. . Such techniques quite common in the art, DNA fragments coding for the are well described. Glycoprotein hormones are then expressed in vivo or in vitro as follows.

  Once a nucleic acid encoding a particular glycoprotein hormone, or a region of the nucleic acid, has been constructed, modified, or isolated, the nucleic acid is then cloned into an appropriate vector, which is wild-type and / or Guides in vivo or in vitro synthesis of modified glycoprotein hormones. The vector is intended to have the functional elements necessary to direct and regulate the transcription of the inserted or hybrid gene. These functional elements include promoters, regions upstream or downstream of the promoter, such as enhancers that can regulate the transcriptional activity of the promoter, origins of replication, suitable restriction sites to aid cloning of the promoter, antibiotics Any other marker, RNA splicing junction, transcription termination region, or anything that helps to promote the expression of the inserted or hybrid gene that helps to select cells containing the substance resistance gene or vector or vector containing the insert Other areas include, but are not limited to (generally see Sambrook et al.).

There are a number of E. coli expression vectors known to those of skill in the art useful for the expression of nucleic acid inserts. Other microbial hosts suitable for use include gonococcal species such as Bacillus subtilis and other Enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, expression vectors can be made that contain expression control sequences that are typically compatible with the host cell (eg, an origin of replication). In addition, there are many well-known various promoters, examples of which include lactose promoter system, tryptophan (Trp) promoter system, β-lactamase promoter system, or promoter system derived from lambda phage. A promoter typically has an operator sequence optionally to control expression, eg, a ribosome binding sequence to initiate and terminate transcription and translation. If required, insertion of an in-frame Met codon 5 ′ to the downstream nucleic acid insert provides an amino terminal methionine. In addition, the carboxy-terminal extension of the nucleic acid insert is removed using standard oligonucleotide mutagenesis methods.

  In addition, yeast expression can be used. There are several advantages to yeast expression systems. First, there is evidence that proteins produced in the yeast secretion system show correct disulfide pairing. Second, post-translational glycosylation is efficiently performed by the yeast secretion system. The Saccharomyces cerevisiae prepro alpha factor leader region (encoded by the MF "-I gene) is commonly used to direct protein secretion from yeast (Brake, et al.," Α-Factor-Directed Synthesis and Secretion of Mature Foreign Proteins in Saccharomyces cerevisiae. "Proc. Nat. Acad. Sci., 81: 4642-4646 (1984)). The leader region of prepro alpha factor is a yeast protease encoded by the signal peptide and the KEX2 gene (this enzyme The nucleic acid coding sequence may be fused in-frame with the pre-pro-α factor leader region. This construct is then a strong transcriptional promoter, such as an alcohol dehydrogenase I promoter or a glycolytic promoter. The nucleic acid coding sequence is followed by a translation termination codon, followed by a transcription termination signal, or the nucleic acid coding sequence can be used to facilitate purification of the fusion protein by affinity chromatography. A secondary protein coding sequence, such as Sj26 or β-galactosidase, is inserted in. The insertion of a protease cleavage site to separate the components of the fusion protein is applicable to constructs used for expression in yeast. Effective post-translational glycosylation and expression of recombinant proteins is also achieved in the baculovirus system.

Mammalian cells allow protein expression in an environment favorable to important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures, and secretion of active proteins. Vectors useful for the expression of active proteins in mammalian cells are characterized by the insertion of protein coding sequences between a strong viral promoter and a polyadenylation signal. Vectors may contain hygromycin resistance, genes that confer gentamicin resistance, or other genes or phenotypes suitable for use as selectable markers, or methotrexate resistance for gene amplification. The chimeric protein coding sequence is introduced into a Chinese hamster ovary (CHO) cell line using a methotrexate resistant coding vector or other cell line using an appropriate selectable marker. The presence of vector DNA in the transformed cells is confirmed by Southern blot analysis. Production of RNA corresponding to the inserted coding sequence is confirmed by Northern blot analysis. Many other suitable host cell lines capable of secreting intact human proteins have been developed in the art, examples of which include CHO cell lines, HeLa cells, myeloma cell lines, Jurkat cells, and the like. Expression vectors for these cells can include expression control sequences such as origins of replication, promoters, enhancers and necessary information processing sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites and transcription terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus and the like. The vector containing the nucleic acid segment is transferred into the host by a well-known method that varies depending on the type of cell host. For example, calcium chloride transformation is commonly utilized for prokaryotic cells, while calcium phosphate, DEAE dextran or lipofectin mediated transfection and electroporation methods are used for other cellular hosts.

  For expression of alternative vectors for expression of genes in mammalian cells, human gamma interferon, tissue plasminogen activator, VIII clotting factor, hepatitis B virus surface antigen, protease Nexinl, and eosinophilic major basic protein The same ones developed in the previous section are used. In addition, the vector can include a CMV promoter sequence and a polyadenylation signal available for expression of the inserted nucleic acid in mammalian cells (eg, COS-7).

  Expression of the gene or hybrid gene is in vivo or in vitro. In vivo synthesis involves transforming prokaryotic or eukaryotic cells that serve as hosts for the vector. Examples of modified glycoprotein hormones that are inserted into prokaryotic expression vectors are given in the Examples section herein.

  Alternatively, gene expression can occur in an in vitro expression system. For example, in vitro transcription systems commonly used to synthesize relatively large amounts of mRNA are commercially available. In such an in vitro transcription system, a nucleic acid encoding a glycoprotein hormone is cloned into an expression vector adjacent to the transcription promoter. For example, the Bluescript II cloning and expression vector contains a multicloning site flanked by a strong prokaryotic transcription promoter (Stratagene Cloning Systems, La Jolla, Calif.). Kits are available that contain all the reagents necessary for in vitro RNA synthesis from a DNA template, such as the Bluescript vector (Stratagene Cloning Systems, La Jolla, CA). The RNA produced in vitro by such a system is then translated in vitro to produce the desired glycoprotein hormone (Stratagene Cloning Systems, La Jolla, CA).

  Another method for producing glycoprotein hormones is to link two peptides or polypeptides together by protein chemistry techniques. For example, a peptide or polypeptide can be prepared using commonly available laboratory equipment using Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonyl) (Applied Biosystems, Inc., Foster City, CA) chemistry. Is synthesized chemically. One skilled in the art can readily appreciate that peptides or polypeptides corresponding to hybrid glycoprotein hormones are synthesized by standard chemical reactions. For example, a peptide or polypeptide is synthesized and not cleaved from its synthetic resin, while the other fragment of the hybrid peptide is synthesized and then cleaved from the resin, thereby functionally blocked on the other fragment. Expose the group. Through the peptide condensation reaction, these two fragments are covalently joined by peptide bonds at their carboxy and amino termini, respectively, to form hybrid peptides. Grant, GA, “Synthetic Peptides: A User Guide,” WHFreeman and Co., NY (1992) and Bodansky, M. and Trost, B., Ed., "Principles of Peptide Synthesis," Springer-Verlag Inc., NY (1993)). Alternatively, peptides or polypeptides are synthesized separately in vivo as described above. Once isolated, these separate peptides or polypeptides are combined to produce a glycoprotein hormone by a similar condensation reaction.

For example, short peptide fragments can be easily ligated to produce large peptide fragments, polypeptides, or whole protein domains by enzymatic ligation of cloned or synthetic peptide segments (Abrahmsen, L., et al., Biochemistry, 30: 4151 (1991)). Alternatively, larger peptides or polypeptides can be constructed synthetically from short peptide fragments utilizing the natural chemical linkage of synthetic peptides. This method consists of a two-step chemical reaction (Dawson, et al., “Synthesis of Proteins by Native Chemical Ligation,” Science, 266: 776-779 (1994)). The first step is a reaction that chemoselectively reacts an unprotected synthetic peptide-α-thioester with another unprotected peptide segment containing an amino-terminal Cys residue to produce a thioester-linked intermediate as an initial covalent product It is. Without changing the reaction conditions, this intermediate undergoes a spontaneous rapid intramolecular reaction to form the original peptide bond at the linking site. Application of this native chemical ligation method to total synthesis of protein molecules is exemplified by the preparation of human interleukin 8 (IL-8) (Clark-Lewis, I., et al., FEBS Lett., 307: 97 (1987), Clark-Lewis, I., et al., J. Biol. Chem., 269: 16075 (1994), Clark-Lewis, I., et al., Biochemistry, 30: 3128 (1991), And Rajarathnam, K., et al., Biochemistry, 29: 1689 (1994)).

  Alternatively, unprotected peptide segments can be chemically linked, in which case the bond formed between the peptide segments as a result of chemical ligation is a non-natural (non-peptide) bond (Schnolzer, M., et al. , Science, 256: 221 (1992)). This technique has been used to synthesize protein domain analogs as well as large quantities of relatively pure proteins with sufficient biological activity (deLisle Milton, RC, et al., “Techniques in Protein Chemistry IV "Academic Press, New York, pp. 257-267 (1992)).

  The present invention further provides fragments of modified glycoprotein hormones having super agonist or antagonist activity. The polypeptide fragment of the present invention is a recombinant protein obtained by cloning a nucleic acid encoding a polypeptide into an expression system capable of producing the polypeptide fragment. For example, the active domain of a glycoprotein hormone can be determined that can interact with a glycoprotein hormone receptor together with the β-subunit to produce a biological effect associated with the glycoprotein hormone. In one example, amino acids that are known not to be involved in glycoprotein hormone activity or binding specificity or affinity are deleted without loss of their respective activities.

  For example, the amino or carboxy terminal amino acid is subsequently removed from the natural or modified glycoprotein hormone and each activity is tested in one of a number of available assays. In another example, a fragment of a modified glycoprotein encloses a modified hormone in which a natural amino acid is replaced with at least one amino acid at a specific position of the α or β-subunit, and a part of the amino-terminal or carboxy-terminal amino acid Or in the internal region of the hormone is replaced with a polypeptide fragment or other moiety that can facilitate the purification of the modified glycoprotein hormone, such as biotin. For example, a modified glycoprotein is fused to a maltose binding protein by peptide chemistry that clones each nucleic acid encoding two polypeptide fragments into an expression vector such that expression of the coding region results in a hybrid polypeptide. The hybrid polypeptide is affinity purified through an amylose affinity column, and the modified glycoprotein is then separated from the maltose binding region by cleaving the hybrid polypeptide with a specific protease factor Xa (eg, New England Biolabs Product Catalog, 1996, pg. 164).

  Active fragments of glycoprotein hormones can be synthesized directly or obtained by chemical or mechanical degradation of larger glycoprotein hormones. An active fragment is an amino acid sequence of at least about 5 contiguous amino acids derived from a natural amino acid sequence and is defined as having an associated activity, such as binding or regulatory activity.

Fragments can be inserted into specific regions or specific amino acid residues as long as the activity of the peptide is not significantly altered or damaged compared to modified glycoprotein hormones, whether or not associated with other sequences. , Deletions, substitutions or other selective modifications. These modifications may provide a number of additional properties such as removing / adding amino acids that can be disulfide bonded or increasing their lifespan. In any case, the peptide must possess bioactive properties such as binding activity, modulation of binding at the binding domain, and the like. The functionality or active region of a glycoprotein hormone can be confirmed by mutagenesis of a specific region of the hormone and subsequent testing of the expression and expression polypeptide. Such methods will be apparent to those skilled in the art, examples of which include mutagenesis of specific sites in the nucleic acid encoding the receptor (Zoller, MJ, et al.).

  In one embodiment of the invention, the human glycoprotein hormone has at least one basic amino acid in the α-subunit at a position selected from the group consisting of positions 11, 13, 14, 16, 17, and 20. In one embodiment, the human glycoprotein hormone has a basic amino acid at position 11. In another embodiment, the human glycoprotein hormone has a basic amino acid at position 13. In another embodiment, the human glycoprotein hormone has a basic amino acid at position 14. In another embodiment, the human glycoprotein hormone has a basic amino acid at position 16. In another embodiment, the human glycoprotein hormone has a basic amino acid at position 17. In another embodiment, the human glycoprotein hormone has a basic amino acid at position 20. In another embodiment of the invention, the basic amino acid at positions 11, 13, 14, 16 and 20 is lysine. In yet another embodiment of the invention, the basic amino acid at position 17 is arginine.

  The present invention further provides a human glycoprotein hormone having a basic amino acid in the α-subunit at the combination of any two positions selected from the group consisting of positions 11, 13, 14, 16, 17 and 20. . For example, a basic amino acid can be at positions 11 and 13, or positions 11 and 14, or positions 11 and 16, or positions 11 and 17, or positions 11 and 20, or positions 13 and 14, or positions 13 and 17, or positions. 14 and 16, or positions 14 and 17, or positions 14 and 20, or positions 16 and 17, or positions 17 and 20. In one embodiment of the invention, the human glycoprotein hormone has basic amino acids at positions 16 and 13. In another embodiment of the invention, the human glycoprotein hormone has basic amino acids at positions 20 and 13. In yet another embodiment, the human glycoprotein hormone has basic amino acids at positions 16 and 20.

  The present invention further provides a human glycoprotein hormone having a basic amino acid in the α-subunit in any combination of three positions selected from the group consisting of positions 11, 13, 14, 16, 17, and 20. . For example, the basic amino acid can be located at positions 11, 13, and 14, or positions 11, 13 and 16, or positions 11, 13 and 17, or positions 11, 13 and 20, or positions 11, 14 and 16, or position 11, 14 and 17, or positions 11, 14 and 20, or positions 11, 16 and 17, or positions 11, 16 and 20, or positions 11, 17 and 20, or positions 13, 14 and 16, or positions 13, 14 and 17, or positions 13, 14 and 20, or positions 13, 16 and 17, or positions 13, 17 and 20, or positions 14, 16 and 17, or positions 14, 16 and 20, or positions 14, 17 and 20. Or at positions 16, 17 and 20. In one preferred embodiment of the invention, the human glycoprotein hormone has basic amino acids at positions 13, 16 and 20. In another embodiment of the invention, the hormone is thyroid stimulating hormone. In another embodiment of the invention, the hormone is a follicle stimulating hormone. In another embodiment of the invention, the hormone is luteinizing hormone. In another embodiment of the invention, the hormone is chorionic gonadotropin. In yet another embodiment of the invention, the basic amino acid at any three positions selected from the group consisting of positions 11, 13, 14, 16, 17, and 20 is lysine.

The present invention further relates to a human thyroid stimulating hormone having at least three basic amino acids in the α-subunit at a position selected from the group consisting of positions 11, 13, 14, 16, 17 and 20, comprising β- Also provided are those having a basic amino acid at at least one position selected from the group consisting of subunit positions 58, 63 and 69. In one embodiment of the invention, the thyroid stimulating hormone has a basic amino acid at position 58 of the β-subunit. In another embodiment of the invention, the thyroid stimulating hormone has a basic amino acid at position 63 of the β-subunit. In another embodiment of the invention, the thyroid stimulating hormone has a basic amino acid at position 69 of the β-subunit. In another embodiment of the invention, the thyroid stimulating hormone has a basic amino acid at each of positions 58, 63 and 69 of the β-subunit. In yet another embodiment of the invention, the basic amino acid at at least one position selected from the group consisting of positions 58, 63 and 69 of the β-subunit is arginine.

  The present invention further provides a human glycoprotein hormone having a basic amino acid in the α-subunit at any combination of any four positions selected from the group consisting of positions 11, 13, 14, 16, 17 and 20. provide. Those skilled in the art will readily determine the possible available combinations. In one embodiment, the human glycoprotein hormone has basic amino acids at positions 11, 13, 16 and 20. In another embodiment, the human glycoprotein hormone has basic amino acids at positions 11, 13, 17 and 20. In another embodiment, the human glycoprotein hormone has basic amino acids at positions 13, 14, 17 and 20. In a preferred embodiment, the human glycoprotein hormone has basic amino acids at positions 13, 14, 16 and 20. In yet another embodiment of the invention, the basic amino acid at any four positions selected from the group consisting of positions 11, 13, 14, 16, 17, and 20 is lysine.

  The present invention further provides a human glycoprotein hormone having a basic amino acid in the α-subunit at any combination of any five positions selected from the group consisting of positions 11, 13, 14, 16, 17 and 20. provide. Those skilled in the art will readily determine the possible available combinations. In one embodiment, the human glycoprotein hormone has basic amino acids at positions 13, 14, 16, 17 and 20. In another embodiment, the human glycoprotein hormone has basic amino acids at positions 11, 13, 14, 16 and 20. In yet another embodiment of the invention, the basic amino acid at any five positions selected from the group consisting of positions 11, 13, 14, 16, 17, and 20 is selected from the group consisting of lysine and arginine. .

  The invention further provides a human glycoprotein hormone having a basic amino acid in the α-subunit at all six positions 11, 13, 14, 16, 17, and 20.

  In another aspect, the invention is a human glycoprotein hormone having a basic amino acid in the α-subunit at at least one position selected from the group consisting of positions 11, 13, 14, 16, 17, and 20. This hormone is a human thyroid stimulating hormone and provides the presence of a basic amino acid at at least one position selected from the group consisting of positions 58, 63 and 69 of the β-subunit. In one embodiment of the invention, the human glycoprotein hormone has a basic amino acid at position 58 of the β-subunit of human thyroid stimulating hormone. In another embodiment of the invention, the human glycoprotein hormone has a basic amino acid at position 63 of the β-subunit of human thyroid stimulating hormone. In another embodiment of the invention, the human glycoprotein hormone has a basic amino acid at position 69 of the β-subunit of human thyroid stimulating hormone. In another embodiment of the invention, the human glycoprotein hormone has a basic amino acid at each of positions 58, 63 and 69 of the β-subunit of human thyroid stimulating hormone. In yet another embodiment of the invention, the basic amino acid at a position selected from the group consisting of positions 58, 63 and 69 is arginine.

  In another aspect, the present invention is a glycoprotein hormone that is human follicle stimulating hormone, human luteinizing hormone or human chorionic gonadotropin, at positions 58, 63 and 69 of the β-subunit of human thyroid stimulating hormone. A corresponding one containing a basic amino acid at at least one position selected from the group consisting of positions in the β-subunit of any glycoprotein hormone is provided. This approach applies equally to non-humans. For example, the β-subunit amino acid sequences of two bovine glycoprotein hormones can be compared and substitutions can be made for any subunit based on sequence differences.

  One skilled in the art can readily determine which sites of the β-subunit of other glycoprotein hormones correspond to positions 58, 63 and 69 of the β-subunit of human thyroid stimulating hormone. See, for example, Ward, et al.,: Bellett, D and Bidard, J. M. (eds) “Structure-function relationships of gonadotropins” Serono Symposium Publications, Raven Press, New York, 65: 1-19 (1990). Here, the amino acid sequences of 26 different glycoprotein hormone β-subunits are aligned and compared. Thus, one skilled in the art can readily substitute non-basic amino acids at these sites of other glycoprotein hormones with basic amino acids.

  Similarly, the invention relates to at least one position selected from the group consisting of a position in the β-subunit of a glycoprotein hormone, wherein the glycoprotein hormone corresponds to the same position of any other human glycoprotein hormone. All human glycoproteins having basic amino acids are provided. For example, comparing the amino acid sequences of the β-subunits of human luteinizing hormone and human chorionic gonadotropin, and based on the amino acid differences between the two β-subunits, the selected site of either of these glycoprotein hormones Can be substituted. This approach applies equally to non-humans.

  The present invention further provides a modified human glycoprotein that exhibits an activity that exceeds that of the wild-type human glycoprotein, and is substituted at a position corresponding to the position of the basic amino acid of the non-human glycoprotein hormone that exhibits an activity that exceeds the wild-type human glycoprotein Provided that contain the same amino acid.

  The non-human glycoprotein hormone having activity over the wild type human glycoprotein can be any non-human species. For example, the non-human species may be bovine. (For example, Benua, RS, et al., “An 18 year study of the use of beef thyrotropin to increase I-131 uptake in metastatic thyroid cancer.” J. Nucl. Med. 5: 796-801 (1964) and Hershman. , JM, et al., “Serum thyrotropin (TSH) levels after thyroid ablation compared with TSH levels after exogenous bovine TSH: implications for I-131 treatment of thyroid carcinoma.” J. Clin. Endocrinol. Metab. (See 1972). Alternatively, the non-human species can be horses, pigs, sheep and the like. In the examples herein, the sequences of 10-21 amino acid regions of 27 species are described.

  The present invention further provides a modified glycoprotein hormone that exhibits an activity over that of a wild-type glycoprotein hormone derived from the same species, and a basic amino acid of a glycoprotein hormone from another species that exhibits an activity over that of a wild-type glycoprotein hormone Those containing the same amino acid substituted at a position corresponding to the position of. Thus, glycoproteins that have been modified to increase their activity are derived from non-human species. For example, comparing porcine glycoprotein hormones with bovine glycoprotein hormones, designing porcine glycoprotein hormones with amino acid substitutions at different positions in the porcine and bovine sequences and constructing porcine glycoprotein hormones with selected changes. The modified porcine glycoprotein hormone can be administered to the pig. Alternatively, the modified glycoprotein may be bovine.

  The present invention further provides a modified glycoprotein hormone having an activity exceeding that of a wild-type glycoprotein hormone derived from the same species, wherein the modified glycoprotein hormone is a different sugar derived from the same species exhibiting an activity exceeding that of the wild-type glycoprotein hormone. What contains the same amino acid substituted in the position corresponding to the position of the basic amino acid of protein hormone is provided. For example, comparing the beta subunits of human thyroid stimulating hormone and human chorionic gonadotropin, amino acid substitutions can be made in any of these beta subunits based on any sequence diversity. Of course, since hormone receptor specificity still needs to be retained, only changes that generally increase or decrease the activity of modified glycoprotein hormones are contemplated. Examples of such β subunit modifications are described in the Examples included herein, where basic amino acids are used for sequence comparisons between human thyroid stimulating hormone and human chorionic gonadotropin. Based on substitutions at positions 58 and 63 of human thyroid stimulating hormone.

  Modification refers to the replacement of a basic amino acid with an abasic amino acid at any particular position in the wild-type glycoprotein. In a preferred embodiment of the invention, these modifications include replacing non-basic amino acids with lysine.

The effect of the modification or modifications on the wild type glycoprotein hormone can be confirmed in a number of ways. For example, cyclic AMP (cAMP) production in cells transfected with modified glycoproteins can be measured and compared to cAMP production of similar cells transfected with wild-type glycoprotein hormones. Alternatively, progesterone production in cells transfected with the modified glycoprotein can be measured and compared to progesterone production in similar cells transfected with the wild-type glycoprotein hormone. Alternatively, it can be measured activity Receptor binding assay of the modified glycoprotein hormone, thymidine incorporation assay, or by T ₄ secretion assay. Specific examples of such assays for measuring the activity of modified glycoprotein hormones are described in the Examples section herein. One skilled in the art can readily determine any suitable assay to use to measure the activity of either wild-type or modified glycoprotein hormones.

  In one embodiment of the invention, the modified glycoprotein hormone has an activity that is at least three times that of the wild-type glycoprotein hormone. This increase in activity is measured by any of the techniques described above and in the examples herein, or in any other suitable assay as readily determined by one skilled in the art. The activity will of course vary and does not have to match from assay to assay or from cell line to cell line. For example, as described in the Examples section herein, the relative potency of the P16K mutation in the α subunit of human glycoprotein hormones is compared to the activity of wild-type glycoprotein hormones in a cAMP assay. , About 6.4 times higher. However, in the progesterone release assay, potency was about 3.4 times and Vmax was 1.6 times. This specific modification is at least about 3-fold active in at least one assay, and thus indicates a glycoprotein having at least 3-fold activity.

  To modify another amino acid position, glycoprotein hormone sequences from human and non-human can be aligned using standard computer software programs such as DNASIS (Hitachi Software Engineering Co. Ltd.). Amino acid residues that differ between human and non-human glycoprotein hormones are then substituted using the techniques described above, and the resulting glycoprotein hormones are assayed using one of the aforementioned assays.

  The subject to be treated or administered a modified glycoprotein hormone can be a human or any non-human mammal. For example, modified glycoprotein hormone superagonists may be used for bovine superovulation by administering these glycoprotein hormones to cows.

  The methods used to replace abasic amino acids at any particular position or positions with basic amino acids can also be used to design glycoprotein hormone antagonists. By making specific substitutions and monitoring the activity of these modified glycoprotein hormones, it can be determined whether the modification produced glycoprotein hormones that exhibit reduced activity. These glycoprotein hormone agonists can be used to test hormone receptors such as receptor turnover rate, receptor affinity for glycoprotein hormones, therapeutic techniques such as the treatment of Graves' disease, and fertility control.

  The present invention further provides a method of treating a condition associated with glycoprotein hormone activity in a subject comprising administering to the subject a therapeutic amount of a glycoprotein hormone of the present invention. These symptoms include any symptom associated with glycoprotein hormone activity. Examples of these symptoms include ovalatory disfunction, luteal phase defect, unexplained infertility, male facter infertility, time-limited conception. However, it is not limited to these.

In another example, glycoprotein hormones can be administered to diagnose and treat thyroid cancer. For example, administration of bovine TSH to human subjects is used to stimulate ¹³¹ I uptake in thyroid tissue to treat thyroid cancer (Meier, CA, et al., “Diagnostic use of Recombinant Human Thyrotropin in Patients with Thyroid Carcinoma (Phase I / II Study). "J. Clin. Endocrinol. Metabol. 78:22 (1994)).

  One of skill in the art can readily determine the effective amount for administration and depending on the weight, size, severity of the particular condition, and the type of subject itself. A therapeutically effective amount can be readily determined by common optimization methods. The present invention provides glycoprotein hormones with increased activity compared to wild type. These modified glycoprotein hormones can achieve similar therapeutic effects at lower doses compared to wild-type glycoprotein hormones, or are therapeutically effective when administered at the same dose as wild-type glycoprotein hormones. An increase can be achieved.

  Depending on whether the glycoprotein hormone is administered orally, parenterally, or by other methods of administration, prostaglandins can be administered in solid, semi-solid or liquid dosage forms such as tablets, pills, capsules, powders, liquids , Creams and suspensions, preferably in unit dosage forms suitable for accurate dosage delivery. A glycoprotein hormone may comprise an effective amount of a selected glycoprotein in combination with a pharmaceutically acceptable carrier, and may further comprise other medicaments, formulations, carriers, adjuvants, diluents, and the like. “Primarily acceptable” means that the substance is not biologically or otherwise undesirable, that is, the substance causes an unacceptable biological action or interacts with a glycoprotein hormone in an unacceptable form. It means that it can be administered to an individual together with a selected glycoprotein hormone without acting. Practical methods for preparing such dosage forms will be known or apparent to those skilled in the art (see, eg, Remington's Pharmaceutical Sciences, latest edition (Mack Publishing Co., Easton, PA)).

  In another aspect, the present invention provides a method of assisting reproduction in a subject comprising administering an auxiliary amount of a glycoprotein hormone of the present invention. For example, in a subject with isolated gonadotropin deficiency (IGD), modified follicle stimulating hormone (folytropin) and luteinizing hormone (lutropin) are administered to the subject to restore normal gonadal function. Glycoprotein hormones, such as FSH and LH, are essential to female reproductive physiology, and these glycoprotein hormones can be administered to subjects to overcome numerous reproductive disorders and thereby assist in reproduction. This is widely known to those skilled in the art.

Gene therapy is another approach to treating hormonal disorders using the modified glycoprotein hormones of the present invention. In this approach, a gene encoding a modified glycoprotein hormone can be introduced into, for example, a germline cell or somatic cell so that the gene is expressed in the cells and subsequent generations of those cells can express the transgene. . For example, any specific gonadotropin can be inserted into ovarian cells, or precursors thereof, to enhance ovulation. Alternatively, when a thyroid cell carrying a gene encoding a thyroid-stimulating hormone superagonist is introduced into an individual having thyroid cancer, the need for continuous administration of TSH to stimulate radioiodine uptake of thyroid cancer is eliminated. Suitable vectors for delivering the coding sequence are well known in the art. For example, the vector is a virus, such as an adenovirus, an adeno-associated virus, a retrovirus or a non-virus, such as a cationic liposome.

  Modified glycoprotein hormones as provided by the present invention may be used for targeted delivery of therapeutic agents to thyroid tissue or gonadal tissue or for the treatment of certain neoplasms.

  In yet another aspect, the present invention provides a method for constructing a superactive non-chimeric analog of a human hormone, comparing the amino acid sequence of a more active homolog from another species with human hormone, and replacing the amino acid in the human hormone with other amino acids. A method comprising the step of substituting the corresponding amino acid from the species and measuring the activity of the substituted human hormone and selecting a superactive analog from the substituted human hormone. Superactive analogs of human hormone include any analog whose activity exceeds the corresponding activity of wild type hormone. For example, modification of human thyroid-stimulating hormone (T11K) from threonine to lysine at position 11 in the α subunit causes a relative increase in cAMP production in JP09 cells cultured in vitro (see herein). See Table II in the examples). Thus, this modification of human thyroid stimulating hormone results in a superactive analog of wild type human thyroid stimulating hormone. As described above, the activity of a homologue from another species is measured, the activity is compared with the activity of a human hormone, the sequenced sequences are then compared to determine amino acid sequence differences, and Replace the amino acid at the corresponding position in the human hormone with the appropriate amino acid in the species-derived hormone, then measure the activity of the modified human hormone by one of the techniques described above, and then determine the activity of the modified human hormone in the wild By selecting superactive analogs from substituted human hormones, as compared to type human hormones, one can determine the particular amino acid or amino acids to be substituted to create a modification.

  All combinations of amino acid substitutions can be used to produce glycoprotein superagonists. For example, neutral amino acids can be substituted with basic or acidic amino acids. Alternatively, basic amino acids can be replaced with acidic or neutral amino acids, or acidic amino acids can be replaced with neutral or basic amino acids. One skilled in the art will recognize that substitution of another amino acid with one amino acid, as described above, may be at the nucleic acid level of a nucleotide sequence encoding a glycoprotein hormone or a portion of a glycoprotein hormone, or at the polypeptide level. Can be recognized. All human hormones are modified by this method and by the superactive analog selected. In particular, the human hormone is a glycoprotein hormone.

The sequence between the human α subunit Cys10 and Pro21 was selected as the primary target for mutagenesis (FIG. 1). hCG-based homology modeling indicates that this region of the α subunit is distant from the β subunit of all glycoprotein hormones, contains several surface exposed residues, and makes one rotation between Pro16 and Ser19 of 3 ₁₀ - including a helix, suggesting that ^1. The human α subunit differs from bovine at positions 11, 13, 16, 17 and 20 (FIG. 1a), and four of these changes (Thr11 → Lys, Gln13 → Lys, Pro16 → Lys and Gln20 → Lys) are Non-conservative. Using PCR amplification, higher apes (chimpanzees-Pan troglodytes, orangutans-Pongo pygmaeus), lower apes (Lena gibbons-Hylobates sp.), Old world monkeys (baboon Papio)
The sequences of the 11-20 regions in several primate α subunits, including anubis), were determined, and they were previously known mammalian sequences such as rhesus monkeys (Macaca mulatta), marmosets (Callithrix jacchus) Compared to New World Monkey) and human (FIG. 1a). Simultaneous comparisons between different species suggested that basic residues in this region were replaced at a relatively late stage in primate evolution. In rhesus α subunit encodes Arg residues Lys residue and position 13 position 11, 16 and 20 ^2, but baboon sequence encodes a Gln in position 16, whereas gibbon sequence WEAK His in position 13 Contains only one basic imidazolium group (FIG. 1a). Apparently, a cluster of positively charged amino acids in this region was retained and modified during vertebrate evolution, but not found in higher apes and human sequences. The gradual exclusion of positively charged residues in the 11-20 region of the α subunit is consistent with the evolutionary branching of the humanoids (human and apes) from the Old World monkey. Our hypothesis that this region could regulate receptor binding was further supported by: 1) highest reactivity to iodination of Tyr21 in bTSH ³ 2) mapping of antigenic determinants in hCG ⁴ 3) acylation of individual subunits ⁵ , labeling ^{6 with} acetic anhydride ⁶ and pegylation The role of the amino group of the sheep and human α subunit Lys for effective hormone-receptor interactions when tested.

  Therefore, a positively charged Lys residue was inserted into the Cys10-Pro21 region of the human α subunit (FIG. 1b). The other two regions were further mutagenized (Table I). A single non-conserved Leu69 → Arg mutation in the TSHβ subunit was made based on similar sequence comparisons.

Effect of mutations Co-transfection into CHO-K1 cells with various combinations of wild-type (WT) or mutant human α and hTSHβ ⁷ or hCGβ cDNA resulted in 14 hTSH and 11 hCG heterodimers. (Table I). In contrast to many other mutagenesis studies ^8,9 , mutant expression was generally comparable to WT. T11K, Q13K, P16K, Q20K, Q50P and Q13K + P16K + Q20K hTSH α mutants were expressed at higher levels than WT-hTSH. Thus, these evolutionarily correct mutations did not significantly impair the synthesis of hTSH or hCG molecules, but in some cases may stimulate hormone production.

Different bioassays were used to compare the relative potency and efficiency of hTSH and hCG mutants. The ability of WT and mutant hTSH to stimulate cAMP production was tested in CHO-JP09 cells with stably transfected human TSH receptors. This assay showed the following order of potency in a single α subunit mutant: P16K (EC ₅₀ of 1/6 of WT) ≧ Q20K>Q13K>T11K> WT−hTSH≈Q50P≈R67K (Table II). The receptor binding activity of WT and mutant hTSH was assessed in a competitive binding assay for porcine thyroid membrane. Consistent with cAMP stimulation, the following order of efficacy was observed. P16K (5 times the affinity of WT)> Q20K ≧ Q13K>T11K> WT−hTSH≈Q50P≈R67K (Table II). Thus, the increased potency of a single mutant observed in JP09 cells directly correlated with increased affinity for the TSH receptor. Most notably, each mutation to a Lys residue in the 11-20 region caused a substantial increase in activity, but changes outside of this critical region indicate receptor binding affinity and biological activity. (R67K, Q50P) had no effect (Table II). Alanine mutagenesis of amino acids 13, 16 and 20 of hTSH did not significantly alter hormonal activity, only that selective re-formation of basic amino acids present in other species of homologous hormones was functional. That caused the change. Furthermore, the replacement of αSer43 with Arg and the replacement of αHis90 and αLys91 indicated that these residues were not as important for hTSH biological activity as for hCG biological activity, indicating that the basic residue hormone Emphasize specific and site-specific roles ⁹ .

Superagonists with combinatorial mutations To further investigate the effects of Lys residues that were separately involved in the highest increase in potency, mutants containing multiple substitutions were made. The most active mutant is shown in FIGS. Double Pro16-> Lys + Gln20-> Lys and triple Pro16-> Lys + Gln20-> Lys + Gln13-> Lys mutants show 12 and 24-fold activity of WT-hTSH, respectively, and up to 35-fold more effective after replacement of Leu69-> Arg in TSHβ subunit Further increased (Fig. 2a). Additional optimization including the Glu14 → Lys substitution (Lys at this position present in the tuna sequence) caused a further increase in biological activity up to 95-fold. These most effective multiple mutants increased potency by at least 1.5 times (maximum response) (FIG. 2b). These increases, testing the ability of hTSH mutants to induce T ₃ production in proliferation as well as cultured human thyroid follicles in FRTL-5 cells bind to porcine as well as human TSH receptor (Fig. 2e) Confirmed by In particular, Pro16-> Lys + Gln20-> Lys + Gln13-> Lys / WT-hTSHβ and Pro16-> Lys + Gln20-> Lys + Gln13-> Lys / Leu69-> Arg mutants to obtain half the maximum stimulation of ³ H-thymidine incorporation in FRTL-5 cells, respectively. Required concentrations of 1/18 and 1/27 of WT-hTSH (FIG. 2e). The synergistic effect of multiple mutations on TSH biological activity was not limited to local cooperation with the receptor for Lys residues in the 13-20 region of the α subunit, but the opposite loop of the β subunit With involvement of Arg69 in (Table II).

These findings were further confirmed in animal models. One injection of Pro16 → Lys, Gln20 → Lys and Gln13 → Lys hTSH mutants in mice increased significantly higher serum T ₄ than WT-hTSH. In addition, Pro16 → Lys + Gln20 → Lys + Gln13 → Lys / WT-hTSHβ and Pro16 → Lys + Gln20 → Lys + Gln13 → Lys / Leu69 → Arg mutant also produced higher T ₄ levels compared to WT-hTSH (FIG. 2f). . The hTSH serum levels at 6 hours after intravenous injection to mice were similar, and the hTSH analogs did not show significant differences in metabolic clearance rates compared to WT.

Sequence comparison of the hCG and hTSH β subunits showed a region containing a cluster of basic residues in hCG (residues 58-69 in TSHβ), but not in hTSH. Using site-directed mutagenesis, single and multiple basic residues were introduced into hTSH based on their position in hCG to create further hTSH β subunit mutants. I58R, E63R, I58R + E63R, I58R + E63R + L69R. Mutant hTSH β subunit is co-expressed with the human α subunit, and the intrinsic activity of the recombinant hTSH analog is expressed in rat THS receptor (FRTL-5 cells) and human TSH receptor (CHO-hTSHr cells). Tested. In both systems, a single substitution (I58R, E63R) increased the potency of hTSH by 2-4 fold, resulting in a slight increase in efficiency (FIG. 2g). The combination of the two substitutions (I58R + E63R) resulted in 15-fold potency and 1.5-fold efficiency of wild type hTSH (FIG. 2g). The ability and efficiency of the combination mutant I58R + E63R + L69R, in which three basic residues were introduced, increased 50-fold and 1.7-fold, respectively (FIG. 2g). These increases in intrinsic activity were accompanied by a concomitant increase in receptor binding affinity as determined in a receptor-binding assay using CHO-JP09 cells (FIG. 2h). Similarly, when mice were injected with the I58R + E63R + L69R mutant, their T ₄ stimulation was significantly higher than sham (MOCK) or control treated mice (FIG. 2i).

The biological activity of CG mutants was tested using progesterone stimulation in MA-10 cells and cAMP stimulation in COS-7 cells transfected with human LH / hCG receptor. The hCG Lys mutant showed higher capacity (low EC ₅₀ value) and higher efficiency (V _max ) than WT-hCG (Table II, FIG. 3a). The effects of single and multiple mutations were relatively similar to those observed for hTSH mutants. The αPro16 → Lys / WT-hCGβ mutant is four times as active as WT-hCG in stimulating progesterone production and receptor binding activity in MA-10 cells, and also Pro16 → Lys + Gln20 → Lys and Pro16 → Lys + Gln20 → It was accompanied by an increase in both capacity and efficiency for the Lys hCG + Gln13 → Lys mutant (FIGS. 3a and 3b). A similar increase in intrinsic activity was also observed when tested with the human LH / hCG receptor (COS-7-hLH / hCG-R cells) (FIGS. 3c and 3d).

  These data suggested that even at higher levels than model hormones (eg bTSH), only a few amino acid substitutions are sufficient to increase glycoprotein hormone biological activity. Interestingly, only 2-3 of the 40 different residues between bovine and human TSH are thought to be responsible for the high biological activity of bTSH. The majority of other substitutions are conserved and appear to have no functional significance, as explained for the R67K mutation at the α subunit. In contrast, it is shown that the surface-localized Lys clustering in the L1 loop and β1 chain of the α subunit is crucial for the high biological activity of bTSH. Thus, recombinant hTSH with only two mutated amino acids (P16K + Q20K) obtains an intrinsic activity comparable to bTSH (Table II). Furthermore, triple, quadruple and quintuple hTSH mutants show even higher capacity than bTSH. These data indicate that the difference in activity between bTSH and hTSH is the result of several amino acid changes, including substitutions that increase activity, but others are the biological capacity of bTSH at the hTSH receptor. This suggests that it can be reduced.

However, similar differences in activity differ, although we cannot rule out the possibility that some receptor species are made from a single transfected cDNA (by alternative splicing from hidden sites or by post-translational modifications) The fact that it has been observed in cell lines strongly counters the importance of different receptor species in increasing the potency, efficiency and affinity of these analogs. Furthermore, natural hormone isoforms with various carbohydrate residues exert their effects at the post-receptor level and have no or minimal effect on receptor binding affinity ¹⁰ . Because wild-type hormones and their analogs are characterized in multiple experimental systems, the phenomenon of increased biological activity described herein is a rule rather than an exception related to specific cell-dependent mutations in the receptor. There is a high possibility that there is.

Conventional site-directed mutagenesis of sight glycoprotein hormone rational design of glycoprotein hormone analogs using strategies such as alanine-scanning mutagenesis ¹¹ or multiple substitution method ^9, the highly conserved regions and residues Mainly focused.
Some important studies have been based on the production of chimeric subunits using cassette mutagenesis and / or restriction fragment exchange ¹² , ¹³ , ¹⁴ . Our strategy based on the substitution of non-conserved residues with those found in other species has been successful, generating other glycoprotein hormone analogs, including hFSH mutants with increased biological activity Made possible. The parallel improvement of the biological activity of hTSH, hCG and hFSH by the introduction of basic residues in the region 11-20 of the human α subunit indicates that this region is in the crystal structure-based model of hCG and our homology of hTSH Related to the fact that there is a distance from the β subunit in the model. The hypothetical identification of this region in both models and the observation ¹⁵ that antibodies that bind to regions 11-26 do not significantly affect subunit combinations suggest that this domain functions similarly in all glycoprotein hormones. . As long as the α subunit is successfully engineered to create a more potent agonist of hTSH, hCG or hFSH, the same paradigm can be used to modify each β subunit, Generate the final super-agonist. For example, the additional substitution of nonpolar Leu69 to Arg in the TSHβ unit caused a further increase in hTSH bioactivity. Furthermore, the plasma half-life of our analogs can be modified for specific therapeutic needs.

Further designs and improvements of glycoprotein hormones include detailed three-dimensional structures of hormone-receptor complexes. The exact structure of the glycoprotein hormone receptor has not been elucidated, but several models of hormone receptor interactions have been proposed ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ . According to the latest model of Jiang et al. ¹⁷ , the L1 loop of the α subunit is involved in the interaction with the transmembrane portion of the receptor. Clusters of positively charged residues within this loop may enhance such interactions and promote further realignment in the receptor, leading to G protein activation and signal transduction.

Materials and Methods Restriction enzymes, DNA markers and other molecular biological reagents were purchased from Gibco BRL (Gaithersburg, MD) or Boehringer-Mannheim (Indianapolis, IN). Cell culture fluid, fetal bovine serum and LipofectAMINE were purchased from Gibco BRL (Gaithersburg, MD). VentR DNA polymerase was purchased from New England Biolabs (Beverly, MA). The full-length human α cDNA (840 bp) and hCG-β gene subcloned into the BamHI / XhoI sites of the pcDNA I / Neo vector (Invitrogen Corp., San Diego, Calif.) H. Obtained from Dr. Ji (University of Wyoming, Laramic, WA). An hTSH-β minigene that did not contain the first intron and had an untranslated first exon and a genuine translation start site was constructed in our laboratory. The rhTSH-G standard is available from Genzyme Corp. (Framingham, MA). CHO cells with stably expressed hTSH receptors (CHO-hTSHR clone JP09 and clone JP26) Offered by Dr. Vassart (University of Brussels, Belgium). Human LH receptor cDNA is described in T.W. Obtained from Dr. Minegishi (Gunma University, Gunma, Japan). FRTL-5 cells are D. Courtesy of Dr. Kohn (NIDDK, NIH, Bethesda, MD). MA-10 cells are This was provided by Dr. Ascoli (University of Iowa, Iowa City, IA). ¹²⁵ I-cAMP and ¹²⁵ I-hTSH were from Hazleton Biologicals (Vienna, VA). Various primate blood samples were obtained from the Yerkes Regional Primate Research Center (Emory University, Atlanta, GA) and Animal Resources (University of Oklahoma, Oklahoma City, OK).

Determination of primate alpha subunit sequence QIAamp ^R blood kit for extraction of genomic DNA from whole blood samples of chimpanzees (Pan troglodytes), orangutans (Pongo pygmaeus), gibbons (Hylobates sp.) And baboons (Papio anubis) (Qiagen Inc., Chatsworth, CA) was used. Genomic DNA was used for PCR. The synthetic oligonucleotide primer used is
5′-CCTGATAGATTGCCCCAGAATGC-3 ′ (sense) (SEQ ID NO: 1) and
5'-GTGATAATAACAAGTACCTGCAGTG-3 '(antisense) (SEQ ID NO: 2)
And synthesized according to the nucleotide sequence of the gene encoding the common α subunit of human glycoprotein hormones ¹⁹ . 800-1000 ng in a 100 μl reaction volume containing 10 mM Tris-HCl (pH 9.0 at 25 ° C.), 50 mM KCl, 2.5 mM MgCl ₂ , 200 μM dNTPs and 2 U Taq DNA polymerase (Promega Corp., Madison, Wis.). PCR was performed using the genomic DNA template and 10 picomoles of each primer. The reaction mixture was covered with mineral oil and each sample was first heated at 95 ° C. for 10 minutes. The PCR program consists of 32 cycles of denaturation at 95 ° C for 1 minute and 30 seconds, annealing at 55 ° C for 1 minute and 30 seconds, and extension at 72 ° C for 1 minute, followed by a final extension period of 7 minutes at 72 ° C. It was done. The reaction was then electrophoresed directly on a 1% agarose gel in the presence of ethidium bromide. Amplified PCR products (˜700 bp) spanning exon 3, intron 3 and exon 4 were purified using the QIAquick PCR generation kit (Qiagen Inc., Chatsworth, Calif.) And the Original TA cloning kit (Invitrogen Corp., San Diego, Calif.). Was subcloned into pCR ^™ II. Fragment sequences were obtained after subcloning or by direct deoxysequencing using a Sequenase kit (US Biochemical Corp., Cleveland, OH).

Homology modeling Modeling is due to strong sequence homology between hCG and hTSH. Cysteine knot residues were aligned as counterparts and the percentage of identical as well as highly conserved substitutions was calculated as described ¹ . The sequence identity between hCG and hTSH was 58%, 31% of the two β subunit sequences were identical, and another 17% contained highly conserved changes in β subunits. The molecular model of hTSH relied on the hCG model template obtained from the crystal coordinates obtained from Brookhaven Data Bank. All coordinate manipulation and energy calculations were performed on Convex using CHARMm release 21.2 and modified using the molecular graph package QUANTA (version 3.3, Molecular Simulation Inc., University of York, York, UK). .

Site-directed mutagenesis The human α-cDNA and hTSHβ minigene were mutagenized by PCR-based megaprimer method ²¹ . Vent ^R DNA polymerase (New England Biolabs, Beverly, MA ) was used to optimize the amplification. After digestion with BamHI and XhoI, the PCR product was ligated into pcDNA I / Neo (Invitrogen Corp., San Diego, Calif.) Using the cleaved BamHI / XhoI fragment. MC106 / p3 E. coli cells were transformed using the Ultracomp E. coli transformation kit (Invitrogen Corp.). A QIAprep 8 plasmid kit (QIAGEN Inc., Chatsworth, Calif.) Was used for multiplex plasmid DNA preparation. Large quantities of plasmid DNA were purified using the QIAGEN Mega and Maxi purification protocols. Multiple mutants were made in the same way using plasmids containing α-cDNA with a single mutation as template for further mutagenesis. Mutations were confirmed by double-stranded sequencing using the Sanger dideoxynucleotide chain termination method.

Recombinant Hormone Expression CHO-K1 cells (ATCC, Rockville, MD) were placed in Ham's F-12 medium containing glutamine and 10% FBS, penicillin (50 units / ml) and streptomycin (50 μg / ml). Maintained. Plate cells (100 mm culture dish) were obtained using p (LB) CMV vector ⁷ or hCGβ- Co-transfected with hTSH minigene inserted in pcDNAI / Neo containing cDNA ⁸ . After 24 hours, the transfected cells were transferred to CHO-serum free medium (CHO-SFM-II, Gibco BRL). Medium containing control medium from mock transfection with expression plasmid containing no gene insert was collected 72 hours after transfection, concentrated and centrifuged. Aliquots were frozen at −20 ° C. and thawed only once before each assay. WT and mutant hTSH were measured and confirmed using four different immunoassays ⁹ as described above. Concentrations of WT and mutant hCG were measured using chemiluminescence assays (hCG kit, Nichols Institute, San Juan Capistrano, CA) and immunoradiometric assays (hCG IRMA, ICN, Costa Mesa, CA).

CAMP stimulation in JP09 cells expressing human TSH receptor CHO cells stably transfected with hTSH receptor cDNA (JP09) were grown and serial dilutions of WT and mutant hTSH as described above ⁹ Incubated together. CAMP released into the medium was measured by radioimmunoassay ²² . An equal amount of total media protein was used as hTSH containing samples from sham controls and transfected cells.

To proliferate transiently transfected treated CHO cells cAMP stimulation hLH receptor cDNA in COS-7 cells expressing human LH receptor, successively similarly WT and mutant hCG and the ²³ essentially Incubated with diluent. CAMP released into the medium was measured by radioimmunoassay ²² . An equal amount of total media protein was used as hCG containing samples from mock controls and transfected cells.

6 hours in assay medium as transformed murine Leydig cells (MA-10) the ²⁴ grown progesterone Stimulating 96 well (well) culture plate in MA-10 cells, together with WT and mutant hCG Incubated. The amount of progesterone released into the medium was measured by radioimmunoassay (CT progesterone kit, ICN Biomedicals, Inc., Costa Mesa, CA).

The receptor binding assay hTSH analogs receptor binding activity, by its ability to displace the ¹²⁵ I-bTSH from solubilized porcine thyroid membranes and assayed ^224. The binding activity of selected analogs with the human TSH receptor was tested using JP09 cells. of hCG analog with MA- cells, and the binding activity of the transiently transfected treated COS-7 cells with the human LH receptor was measured using the ¹²⁵ I-hCG and assay medium as described above ^24.

Proliferation thymidine incorporation stimulated rat thyroid cells in FRTL-5 cells (FRTL-5), it was monitored as described above ^22.

The in vivo biological activity of stimulating WT and mutant TSH of T ₄ secretion in mice was measured using a modification of the bioassay ^{22 and 25} of MacKenzie. WT and mutant TSH were administered intraperitoneally into male albino Swiss Cr1: CF-1 mice that had previously suppressed endogenous TSH by administering ₃ μg / ml T ₃ in drinking water for 6 days. After 6 hours, blood samples were taken from the orbital sinus and serum T ₄ and TSH levels were measured by chemiluminescence assay (Nichols Institute), respectively.

Throughout this application, various publications have been referenced. Some publications are numbered in parentheses. All references are numbered and listed below. In order to describe in more detail the state of the field to which the present invention pertains, these descriptions are incorporated herein by reference.

  It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the scope and spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed in this application. The description and examples are for illustrative purposes only, and the true scope and spirit of the invention is intended to be indicated by the following claims.

FIG. 1 shows a comparison of related primary sequences of α-subunits from 27 different species. (A) Subunit sequences obtained from sequencing of PCR amplified fragments of chimpanzee, orangutan, gibbon and baboon (underlined) genomic DNA obtained from GenBank, SWISS-PROT and PDB databanks were aligned. The sequence number corresponds to that of the human α-subunit sequence. The horizontal line (---) indicates the same amino acid residue as that of the human α-subunit. Conserved lysine residues between different species are shown in bold. The primate sequences determined in this study are underlined. The α-subunit sequences of human, chimpanzee and orangutans are sequences that do not contain basic amino acids in this region, despite the relatively high degree of similarity in various vertebrate species. (B) Human sequence mutations made in this region included the introduction of single and multiple Lys residues present in all non-human mammalian sequences. In addition, mutations of residues 13, 16 and 20 to alanine were used to investigate the role of Gln13, Pro16 and Gln20. FIG. 2 shows the biological activity and receptor binding activity of the most potent hTSH analogs. (A, b) cAMP stimulation in CHO-JP09 cells. Data represent the mean ± SEM of triplicate measurements from a representative experiment repeated 3 times (a) and 2 times (b). FIG. 2 shows the biological activity and receptor binding activity of the most potent hTSH analogs. (A, b) cAMP stimulation in CHO-JP09 cells. Data represent the mean ± SEM of triplicate measurements from a representative experiment repeated 3 times (a) and 2 times (b). FIG. 2 shows the biological activity and receptor binding activity of the most potent hTSH analogs. (C, d) Receptor binding activity against CHO-JP09 cells. Mutants identical to those in FIGS. 2a and 2b, respectively, were tested. Values are mean ± SEM of quadruplicate measurements from one experiment repeated twice. FIG. 2 shows the biological activity and receptor binding activity of the most potent hTSH analogs. (C, d) Receptor binding activity against CHO-JP09 cells. Mutants identical to those in FIGS. 2a and 2b, respectively, were tested. Values are mean ± SEM of quadruplicate measurements from one experiment repeated twice. FIG. 2 shows the biological activity and receptor binding activity of the most potent hTSH analogs. (E) Stimulation of thymidine incorporation in FRTL-5 cells. Values are mean ± SEM of quadruplicate measurements from one experiment repeated twice. FIG. 2 shows the biological activity and receptor binding activity of the most potent hTSH analogs. (F) stimulation of T ₄ secretion in mice. Each data point represents the mean ± SEM of values from 4-5 animals in each experiment repeated twice. FIG. 2 shows the biological activity and receptor binding activity of the most potent hTSH analogs. (G) cAMP stimulation in CHO-hTSH cells. Data represent the mean ± SEM of 3-4 replicates from each experiment repeated three times. FIG. 2 shows the biological activity and receptor binding activity of the most potent hTSH analogs. (H) Receptor binding activity in CHO-JP09 cells. Data represent the mean ± SEM of 3-4 replicates from each experiment repeated three times. FIG. 2 shows the biological activity and receptor binding activity of the most potent hTSH analogs. (I) stimulation of T ₄ secretion in mice. Each data point represents the mean ± SEM of values from 4-5 animals in each experiment repeated twice. FIG. 3 shows the biological and receptor binding activities of the most potent hCG analogs. Progesterone production stimulation in MA-10 cells (a) and receptor binding assay (b). Data represent the mean ± SEM of triplicate measurements from each experiment repeated three times. The relative maximum production level of progesterone is shown in Table II as a percentage of the value obtained with WT-hCG. FIG. 3 shows the biological and receptor binding activities of the most potent hCG analogs. Progesterone production stimulation in MA-10 cells (a) and receptor binding assay (b). Data represent the mean ± SEM of triplicate measurements from each experiment repeated three times. The relative maximum production level of progesterone is shown in Table II as a percentage of the value obtained with WT-hCG. FIG. 3 shows the biological and receptor binding activities of the most potent hCG analogs. cAMP stimulation (c) and receptor binding assay (d) in COS-7 cells expressing the hLH receptor. Data represent the mean ± SEM of triplicate measurements from each experiment repeated twice. FIG. 3 shows the biological and receptor binding activities of the most potent hCG analogs. cAMP stimulation (c) and receptor binding assay (d) in COS-7 cells expressing the hLH receptor. Data represent the mean ± SEM of triplicate measurements from each experiment repeated twice.

Claims (32)

A wild-type human chorionic gonadotropin hormone different modified human chorionic gonadotropin (CG), the modified human C G contains α- subunit and β- subunits, the α- subunit position A modified human CG comprising at least 3 basic amino acids at a position selected from the group consisting of 11 , 13 , 16, and 20 . The modified human CG of claim 1, wherein the basic amino acid of the α-subunit is present at positions 11, 13 and 20 . The modified human CG of claim 1, wherein the basic amino acid of the α-subunit is present at positions 13, 16 and 20 . The modified human CG of claim 1, wherein the basic amino acid of the α-subunit is present at positions 11, 13, 16 and 20 . The modified human CG according to any one of claims 1 to 4 , wherein the basic amino acid is selected from the group consisting of lysine and arginine . A nucleic acid encoding the α-subunit of the modified human CG according to any one of claims 1 to 5 . A vector comprising the nucleic acid of claim 6 . A host comprising the vector of claim 7 . The modified human CG according to any one of claims 1 to 5 , further having less than 5 amino acid substitutions at positions other than positions 11 , 13 , 16, and 20 of the α-subunit . The modified human CG according to any one of claims 1 to 5 , further having less than 4 amino acid substitutions at positions other than positions 11 , 13 , 16, and 20 of the α-subunit . The modified human CG according to any one of claims 1 to 5 , further comprising less than 3 amino acid substitutions at positions other than positions 11 , 13 , 16, and 20 of the α-subunit . The modified human CG according to any one of claims 1 to 5 , further comprising less than 2 amino acid substitutions at positions other than positions 11 , 13 , 16, and 20 of the α-subunit . α- subunit position 11, 13, 1 6 and any one of claims 1 to 5 having an amino acid sequence identical to wild-type human C G corresponding to the position other than the 20 modified human C G. A wild-type human chorionic gonadotropin hormone different modified human chorionic gonadotropin (CG), the modified human C G contains α- subunit and β- subunits, the α- subunit position A modified human CG comprising a basic amino acid at at least one position selected from the group consisting of 11 , 13 , 16, and 20 . The modified human CG of claim 14 , wherein the basic amino acid of the α-subunit is at position 11 . The modified human CG of claim 14 , wherein the basic amino acid of the α-subunit is present at position 13 . modification according to claim 14, basic amino acid α- subunit is present at position 16 the human C G. The modified human CG of claim 14 , wherein the basic amino acid of the α-subunit is at position 20 . The modified human CG according to any one of claims 14 to 18 , wherein the basic amino acid is selected from the group consisting of lysine and arginine . 15. The modified human CG of claim 14 , wherein the α-subunit is further modified to contain a basic amino acid at at least two positions selected from the group consisting of positions 11 , 13 , 16, and 20 . of claim 20, basic amino acid α- subunit is present in position 16 and 20 modified human C G. 21. The modified human CG of claim 20 , wherein the basic amino acid of the α-subunit is present at positions 16 and 13 . of claim 20, basic amino acid α- subunit is present in position 20 and 13 modified human C G. 24. The modified human CG of any one of claims 20 to 23 , wherein the basic amino acid is selected from the group consisting of lysine and arginine . A nucleic acid encoding the α-subunit of the modified human CG according to any one of claims 14 to 24 . A vector comprising the nucleic acid of claim 25 . A host comprising the vector of claim 26 . Further α- subunit positions 11, 13, 1 6 and any one of claims 14 to 24 having less than five amino acid substitutions at positions other than the 20 modified human C G. The modified human CG according to any one of claims 14 to 24 , further comprising less than 4 amino acid substitutions at positions other than positions 11 , 13 , 16, and 20 of the α-subunit . Further α- subunit positions 11, 13, 1 6 and any one of claims 14 to 24 having less than three amino acid substitutions at positions other than the 20 modified human C G. 25. The modified human CG of any one of claims 14 to 24 , further comprising less than 2 amino acid substitutions at positions other than positions 11 , 13 , 16, and 20 of the α-subunit . α- subunit position 11, 13, 1 6 and any one of claims 14 to 24 having an amino acid sequence identical to wild-type human C G corresponding to the position other than the 20 modified human C G.

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