JPH09505741A - Single-chain form of glycoprotein hormone quartet - Google Patents

Abstract

  (57) [Summary] Disclosed are single-chain forms of the glycoprotein hormone quartet, at least some members of which are found in most vertebrates. In one embodiment of these single chain forms, the α and β subunits of the wild-type heterodimer or variant thereof are covalently linked, optionally via a linker moiety. Drugs can be included in the linker moiety targeted to the receptors for these hormones. Some of the single chain forms are agonists of glycoprotein hormone activity, and others are antagonists. Another embodiment of the single chain compound of the present invention comprises two β subunits of a glycoprotein hormone, the β subunits being the same or different. These “2-β” forms are antagonists of glycoprotein hormone activity.

Description

Detailed Description of the Invention                Single-chain form of glycoprotein hormone quartetDescription of government assistance   This invention was funded by the National Institutes of Health under NIH contract number NO1-HD-9-2922. It was made with government support. The government has certain rights in this invention.Technical field   The present invention is directed to protein engineering and glycoproteins that naturally occur as heterodimers. The field of protein hormones. In particular, the present invention relates to chorionic gonadotropin (CG ), Thyroid-stimulating hormone (TSK), luteinizing hormone (LH), and follicle-stimulating hormone. Relates to the single-chain form of Lumon (FSH).Background technology   Four important glycoprotein hormone heterodimers (LH, FSH) in humans , TSH, and CG) have the same α subunit and different β subunits. . Three of these hormones are also present in virtually all other vertebrate species, CG To date, it has only been found in primates and the horse placenta and urine.   PCT Application No. WO9, published September 7, 1990 and incorporated herein by reference 0/09800 describes many modified forms of these hormones. One important Modification of the carboxy-terminal peptide of human chorionic gonadotropin or its variants. C-terminal extension of the β subunit by the peptide. Others of these hormones Muteins of E. coli have also been described. The relevant location of CTP is human chorionic gonadal stab From the arbitrary position of 112-118 of the super hormone β subunit to the 145th position. PCT The application is for a preserved film that does not destroy the ability of CTP to alter clearance properties. The mutants of CTP elongation obtained by acid substitution are described. Further, in this specification US Application No. 08 / 049,869 filed April 20, 1993, incorporated by reference No. is for these hormones due to the extension or insertion of CTP at a position other than the C-terminus. And a CTP fragment shorter than the sequence spanning positions 112-118 to 145. It is listed.   The CTP-expanded β subunit of FSH is also described in the applicant's two reports herein. Featured: LaPolt, P.S., et al.Endocrinology(1992)131: 2514-2520 and Fa res, F.A. et al .;Proc Natl Acad Sci USA(1992)89: 4304-4308. Both of these reports , Incorporated herein by reference.   The crystal structure of the heterodimeric form of human chorionic gonadotropin is about the same time. Published in a paper by Lapthorn, A.J., et al.Nature(1994)369: 455-461 And the other is Wu, H. et al.Structure(1994)2: 545-558. these The paper results are from Patel, D.J.Nature(1994)369: 438-439.   At least one example of the preparation of good single-stranded forms of heterodimers is known. Monellin, a naturally occurring sweet protein, is a heterodimeric form of selenium. Isolated from dipitty berries. To investigate the crystal structure of heterodimers , The spatial characteristics of the heterodimer form are maintained, and the C-terminus of the B chain is linked to the N-terminus of the A chain. This is consistent with the fact that they can be connected via an anchor. Such ligation results in sweet protein It provides this molecule in a stable form at high temperatures for use as a quality It is useful because it is convenient for Described in US Pat.No. 5,264,558 This has been successfully achieved by preparing single-stranded forms, as Therefore, heat denaturation was hindered.   PCT application WO 91/16922, published on November 14, 1991, describes many chimeric and A modified form of the heterodimeric glycoprotein hormone is described. Generally, this The disclosures are α subunits, each containing various α or β chain moieties. Focuses on β-subunit chimeras. Simply posted in this application One construct that is only described but not otherwise specifically described is human chorionic gonadotropin. Substantially all of the β chain of the hormone is the α subunit preprotein (ie, Containing the secretory signal sequence of the subunit of β chain and α chain The presence of an intervening signal sequence is defined and described herein. This construct is outside the scope of the invention as it does not act as such a linker moiety.   Ordinary heterodimeric glycoprotein hormones have various CTP extensions and Retains their properties when in single-stranded form, including single-stranded forms that contain insertions Was recognized.Disclosure of the invention   The present invention provides single chain forms of glycoprotein hormones. Low in this hormone Some, if not some, are found in most vertebrate species. Of the present invention The single-chain form may be glycosylated, partially glycosylated, or non-glycosylated. Α that is present in naturally occurring glycoprotein hormones or variants thereof The chain and β chain (or α and α, or β and β) may be a linker part, if necessary. Can be connected via minutes. Particularly preferred linker moieties are complete units or Are only those parts, and the carboxy terminal peptide (carboxy terminal peptide; CTP) unit. The obtained single-chain hormone is an unmodified heterodimer form Retain the activity of or are antagonists of this activity.   Thus, in one aspect, the invention provides for the amino acid modification of one β subunit of the hormone. Sugar chain covalently linked to the amino acid sequence, optionally via a linker moiety. The amino acid sequence of the α subunit common to protein hormones or their amino acids Contains a variant of the no acid sequence, where the variant is defined herein. , Glycosylated or non-glycosylated proteins.   In another aspect, the invention provides the amino acid sequence of one β subunit of the hormone. To the glycoprotein group, which is covalently linked via a linker moiety as necessary. The amino acid sequence of the β subunit of the member of the Lumon Quartet (Quadruple), Or, variants of those amino acid sequences (where variants are defined herein). ) Containing glycosylated or non-glycosylated proteins Have been.   In another aspect, the present invention relates to the amino acid sequence of the other α subunit, optionally And the glycoprotein hormone chart covalently linked via a linker moiety. Amino acid sequence of the α subunit of G. or mutants of these amino acid sequences ( Wherein the variant is a glycosylation containing (as defined herein) Or non-glycosylated proteins.   In yet another aspect, the invention provides that the efficacy is a single-chain form of the hormone quartet. More Predicted Biologically Important Dimeric Glycosylation or Non-Glycosylation It is directed to the single-stranded form. Therefore, the present invention also includes interleukin 3 and And 12 (IL-3 and IL-12), tumor necrosis factor (TNF), transforming growth It is directed against the factor (TGF), and the single-chain form of inhibin. One derived from IL-3 Like a single-stranded form of one subunit and the other subunit from IL-12 , Hybrid interleukins are also included.   In another aspect, the invention provides a set for producing a single chain protein of the invention. Alternative materials and methods, pharmaceutical compositions containing them, antibodies specific for them, And directed to their use.Brief description of the drawings   Figure 1 shows the fusion of the third exon of CGβ with the second exon encoding the α subunit. 2 shows the construction of SalI restricted DNA fragment.   Figure 2 shows the amino acid sequence of human CGβ and the numbering at positions 112-145.   Figure 3 shows the results of competitive binding assay of FSH receptor with various FSH analogs. Is shown.   Figure 4 shows the signal transduction assay for the FSH receptor for various FSH analogs. The result of the say is shown.MODE FOR CARRYING OUT THE INVENTION   The four human "glycoprotein" hormones are human chorionic gonadotropin (hC G), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and thyroid-stimulating hormone. Provides a family that includes Lumon (TSH). As used herein , "Glycoprotein hormones" are found in humans and other vertebrates It is a member of the ruko family. All of these hormones are specific species For the α subunit, which has the same amino acid sequence in the group, and Heterodimers composed of different β subunits according to Millie members It is. Therefore, these glycoprotein hormones are usually associated with each other. Is composed of α and β subunits that are not covalently bound to Exists as a rhodimer. Most vertebrates produce FSH, TSH, and LH However, chorionic gonadotropin is found only in primates, including humans, and horses . A particular form of CG from horses is called pregnant horse serum glycoprotein (PMSG).   Therefore, this hormone "quartet (quadruplet)" is And β subunit are encoded by different genes and synthesized separately by the host. It is composed of heterodimers. Next, the host can The knit is assembled into a non-covalently linked heterodimer complex. this Thus, this hormone quartet heterodimer is composed of a single gene. (In this case by intervening “pro” sequences), subunits Insulin-like hete, covalently coupled by a Ruffido bond Different from Lodimer. This hormone quartet is also assembled from different positions. Immunoglobules that stand up but are covalently linked by disulfide bonds Is also distinguished. However, on the other hand, the plant protein monerin And are held together by non-covalent interactions between the B and B chains. Present time In point, it is not known whether the two chains are encoded on separate genes.   Therefore, various factors may be formed by the same or different subunits. Affects the determination of the action of biologically active compounds that are immers. The subunit is Can be covalently or non-covalently linked and can be linked by the same or different genes. Can be synthesized by the method described above and, in the precursor form, joins the two members of the dimer. B) may or may not contain sequences. Glycoprotein forms herein Based on the results obtained with the single-stranded form of the monquartet, it is biologically active Dimers interleukin-12 and interleukin-3 (IL-12 and IL-3), inhibin, tumor necrosis factor (TNF), and transforming The single chain form of long factor (TGF) is also apparent to be biologically active.   The single-stranded forms of heterodimers or homodimers are their dimeric forms. It has many advantages over. First, they are generally more stable. Especially LH Is noted for instability and short half-life. Second, only one gene is transferred The problem of recombinant production is reduced as it has to be copied, translated and processed. You. This is especially important for bacterial expression. Third, of course, They provide alternative forms that allow for fine tuning of activity levels and half-life in vivo. Can be adjusted. Finally, the single-stranded form identifies a truncated form with dimeric activity. It is a unique starting material for Due to the linkage between subunits, all proteins are folded Proteins can be engineered to be produced without perturbing folding.Characteristics of quartet members   The β subunit of hCG is substantially larger than the other β subunits, and The subunit is about 34 additional amino acids at the C-terminus (herein the carboxy-terminal portion). (Called a carboxy terminal portion; CTP), and this is an O-bond Due to this extension, other gonadotropins, when glycosylated at the site The serum half-life of hCG is believed to be relatively long compared to that of (Matzuk, M. et al., E. ndocrinol (1989)126: 376). In natural hormones, this CTP extension is Containing O-linked oligosaccharides.   In one embodiment of the invention, the α and β chains of the glycoprotein hormone are labeled. And the α and β chains are covalently linked, optionally via a linker moiety. It becomes a single-stranded proteinaceous substance linked by a bond. The linker part is A CTP unit as described herein may include the amino acid sequence Can be advantageously included in the vehicle. In addition, the linker targets the hormone receptor It can include peptide or non-peptide drugs that can be attached.   By simply coupling the two peptide chains via a peptide bond In addition to the head-to-tail arrangement achieved, the α and β chains are head-to-head ( head-to-head or tail-to-tail. Head-to-head and Tail-to-tail coupling involves coupling two carboxyl groups or Includes synthetic chemistry using standard methods of linking two amino groups. For example , By standard activation methods, the two amino groups are It can be linked via any dicarboxylic acid derivative; the two carboxyl groups are It can be linked via a min or a diol. However, the most preferred form is the head -Tail configuration, where standard peptide bonds are sufficient and single-stranded The compound may be recombinantly fused as a fusion protein or in a single chain. Or preferably by synthetic peptide methods linking individual parts of the entire sequence. Can be done. Of course, if desired, peptide or non-peptide linker moieties Minutes can be used in this case as well, but this is not required and The advantage of recombinant production of this chain protein is that the embodiment enabling this production method is It is suggested to include the most preferred approach.   When a head-to-tail configuration is used, the linker consists essentially of the added peptide sequence. Can be As with the heterodimers, the two β chains are described further below. Can be linked via a CTP unit. Therefore, possible implementations of the invention Embodiments include α-FSHβ, βFSH-α, α-βLH, α-CTP-FSHβ, which have an N-terminal on the left. βLH-CTP-α, CTP-βLH-CTP-α and the like are included.   Single-chain form of heterodimeric gonadotropin or glycoprotein quartet The embodiment also relates to a further important set of embodiments, in this embodiment the α sub Instead of coupling the unit with the β subunit, two β subunits Or the two α subunits are grouped together to form a single-chain compound. Can be pulled. As with heterodimers, binding is head-to-head, head-to-head. It can be tail or tail-to-tail.   "2-β" single-chain tandem peptide is a heterodimer glycoprotein hormone Therefore, it is useful as an antagonist to a normally activated receptor. The β subunit confers receptor specificity, while the α subunit transduces a signal Of the α and β subunits are The single-chain compound binds specifically to the receptor because it has a similar conformation. Should be able to bind to each other so that at least one β chain activates the receptor. It exists without being transformed.   The antagonistic activity of "2-β" single-chain tandem peptides is Based in part on the crystal structure. The α and β chains have the same cystine knot configuration , And some folding patterns of the two chains are noted to be similar.   The “2-β” single-chain compound of the present invention is a tandem copy of the same β subunit. That is, FSHβ-FSHβ; HCGβ-HCGβ; TSHβ-TSHβ; or LHβ-LHβ) Or HCGβ-FSHβ; FSHβ-LHβ; LHβ-TSHβ. Any chimeric single chain compound can be used. Such possible combinations are the total There are 12 of them. In addition, the carboxyl-terminal peptide of the HCG β subunit (CTP ), When present between the two β-strands, causes the conformation of the single-stranded compound. Improve. This occurs automatically when HCGβ is the upstream part. But that In other examples, the carboxy of those involved as upstream, as described herein. It is advantageous to use the CTP subunit at the end of the loop. Two such CTP users Knits are also within the scope of the invention. Therefore, a preferred embodiment is FSHβ-CTP- Includes FSHβ; FSHβ-CTP-CTP-FSHβ; LHβ-CTP-LHβ; LHβ-CTP-CTP-FSHβ You.   Similar statements apply to "2α" single chain compounds, of course, α variants Chimeric pairs other than those related to are not included. Various linkers (preferably CTP And CTP extension are also included.   The following definitions can help describe the single-stranded form of the molecule.   As used herein, the α subunit, and FSH, LH, TSH, and And CGβ subunits, as well as heterodimeric forms, are generally Meaningful and known per se in the art, regardless of the glycosylation pattern exhibited. Having the amino acid sequence of, or allelic variants thereof It is.   The "native" forms of these peptides are amino acids isolated from the relevant vertebrate tissues. Has a non-acid sequence, which itself has a known sequence or allelic variants thereof Peptide.   “Mutant” forms of these proteins are, for example, site-directed mutagenesis or Meanings in the amino acid sequence of naturally occurring proteins produced by other recombinant manipulations A protein with schematic modifications or a synthetically prepared protein is there.   These modifications include deletions, insertions and substitutions, 1-10, preferably 1-8, More preferably 1-5 amino acid changes, most preferably defined below. Consists of such conservative amino acid substitutions. The resulting mutant has a corresponding activity to the natural hormone. Retains the effecting activity, that is, directly retains the biological activity of the natural hormone Must be or can bind to receptors for natural hormones in general Does not act as an antagonist by lacking the ability to affect signal transduction I have to. For example, the glycosylation site at position 52 of the α subunit is Removed by acid substitution, which prevents total glycosylation at that site , A hormone that is a heterodimer having this modified α subunit is generally A It is a gonist that binds to receptors and competes for the binding of natural hormones. Can be blocked. (On the other hand, the glycosylation site of the α subunit at position 78 It seems to have little effect on activity. ) Other modifications in the amino acid sequence are also , May produce antagonistic activity rather than agonistic activity for the variant.   One set of preferred variants contains either α or β subunits or Is a variant in which both glycosylation sites have been modified. α subunit is 2 Contains one glycosylation site, one at position 52 and the other at position 78 for activity The effect of modification of these sites is just described. Similarly, β The subunit contains two N-linked glycosylation sites (depending on the nature of the β chain, Similar alterations may be made at these sites). CCG extension of hCG It contains four O-linked glycosylation sites and contains conservative mutations at serine residues (eg, serine residues). Conversion to alanine) destroys these sites. O-linked glycosylation moiety Disruption of position affects the conversion of agonist activity to antagonist activity. It can sound.   Finally, the amino acid sequence adjacent to the N-linked or O-linked glycosylation site. Mutations in the resulting molecule affect the glycosylation properties present on the resulting molecule and make it more active. To change.   Modifications in the amino acid sequence also include both insertions and deletions. Therefore, A truncated form of rumone, eg, at the C-terminus, some or all of amino acids 85-92. Variants of the α subunit lacking no acid are included in the variant. further, Included are alpha subunits with 1-10 amino acids deleted from the N-terminus. In this specification Some useful variants of the described hormonal quartet were published on January 5, 1993. No. 5,177,193 issued to U.S. Pat. Is used as. Glycosylation patterns, as shown in that patent Disrupts relevant sites or, alternatively, selects the host in which the protein is produced. Therefore, it can be modified.   As explained above, the single-chain form is associated with various engineered mutant proteins. It is a convenient starting material. Such muteins may be modified or removed. Includes muteins with non-critical regions that are deleted. this Such deletions and modifications can include complete loops and consist of more than 10 amino acids The sequence may be deleted or modified. However, single-stranded molecules are -Must retain the binding domain and / or the region associated with signal transduction No.   Important Literature on Hormonal Quartet Variants Described Here Is present and, from this reference, produces both agonist and antagonist activity It is clear that many possible variants can be prepared. Such variants are For example, Chen, F. et al.Molec Endocrinol(1992)6: 914-919; Yoo, J. et al.,J Biol Chem (1993)268: 13034-13042; Yoo, J. et al.,J Biol Chem(1991)266: 17741-17 743; Puett, D. et al.Glycoprotein Hormones, Lusbader, J.W. et al., EDS,Springer  Verlag New York (1994) 122-134; Kuetmann, H.T. et al., (Ibid.) 103-117; Er. ickson, L.D. et al.Endocrinology(1990) 126: 2555-2560; and Bielinska, M. et al. ,J Cell Biol(1990)111: 330a (Abstract 1844).   As described above, one method of constructing effective antagonists is to It contains two β subunits of the same or different members of the protein quartet. To prepare a single-stranded molecule. Particularly preferred variants of these single chain forms Is one or more cysteine bonds, typically one or both involved in the bond. By replacing the cysteine with the neutral amino acid Including. A particularly preferred cysteine bond that can be deleted is between position 26 and 110, and And a bond between positions 23 and 72.   Furthermore, the β subunit of the hormone quartet is the biological component of both components of the chimera. To provide a hormone of functional or, in general, altered biological function As such, it has been demonstrated that it can be constructed in a chimeric form. Therefore, of FSH and LH / CG Chimeric molecules exhibiting both activities are Moyle,Proc Natl Acad Sci(1991)88: 760-764; M oyle,Nature(1994)368: 251-255. These reports In the amino acids 101-109 of FSH-β and in the CG-β subunit as disclosed in Substitution with the corresponding residue in ss yields analogs with both hCG and FSH activity .   These chimeric forms of the β subunit also couple the two β subunits. It can be used in single chain compounds that are ringed into one molecule.   Glycosylation patterns have a significant impact on activity qualitatively and quantitatively. It is recognized that, for convenience, the terms FSH, LH, TSK and CGβ subunits Is the amino acid sequence characteristic of the peptide, such as for the "alpha subunit". It is a row. When only the β chain is mentioned, the term is, for example, FSHβ. And the simple term "FSH" is used when referring to heterodimers. You. The glycosylation pattern can be, for example, a recombinant expression host or glycosylation site. It is clear from the background how it is affected by position mutations . Of note are glycoprotein forms that have a particular glycosylation pattern.   As used herein, "peptide" and "protein" refer to The length distinction between them is arbitrary and is used interchangeably.   In the single-stranded form of the invention, the α and / or β chains are inserted in a non-critical region CTP extension.   The “non-critical” regions of the α and β subunits are biologically active (agonism). This is a region of the molecule that is not required for striking and antagonist activity). General In general, these regions separate from binding sites, precursor cleavage sites, and catalytic sites. Is done. Inhibition of proper folding, binding to receptors, catalytic activity, etc. Areas of interest should be avoided, as well as the three-dimensional conformation of proteins. Areas that are important for securing security should be avoided. Heavy for dimers Some areas that are important are the conformational constraints imposed by single strands. In single-stranded form, as the restriction can eliminate the need for these regions. It should be noted that Confirmation of non-important region is deletion of candidate region Or by modification and performing appropriate assays for the desired activity. Will be completed. Regions where the modification causes loss of activity are important and may be the same Or regions that produce similar activity (including antagonist activity) are considered to be insignificant available.   By “biological activity”, an agonist or It is emphasized that an activity that is antagonistic is meant. Therefore, Antago Although nysts cannot directly provide the physiological effects of hormones, certain areas are Important for the action of the mutant as a tagonist.   For example, for the α subunit, positions 33-59 are required for signal transduction. Possible and carboxy-terminal 20 amino acid sequence is signal transduction / receptor Required for binding. Residues important for assembly with the β subunit are at least 33 -58 residues, especially 37-40 residues.   If the non-critical region is "close" to the N- or C-terminus, the insertion is Any position within the mino acid, preferably within 5 amino acids, and most preferred Ku is at the end itself.   Generally, "proximal" means within 10 amino acids of the subject position, preferably 5 amino acids. Used to indicate a location within acid, and most preferably the location of interest itself It is. Thus, certain variants will have amino acid positions "close" to the glycosylation site. Exchange may be included. The definition here relates to this. In addition, the α subunit and Beta subunits are in close proximity to each other at their N- or C-termini. Can be linked to.   As used herein, a "CTP unit" refers to amino acids 112-118. To chorionic C-terminal residue 145, or extending to that part, human chorionicity This is the amino acid sequence found at the carboxy terminus of the gonadotropin β subunit. And. Therefore, each "complete" CTP unit depends on the N-terminus of CTP, depending on the 28-34 Contains 1 amino acid. The native sequence at positions 112-145 is shown in Figure 2.   Exists from 112-118 to 145, depending on the "partial" CTP unit But from the shortest possible “complete” CTP unit (ie, from positions 118-145) An amino acid sequence with at least one amino acid deleted is meant. Included in the present invention The “partial” CTP unit is preferably, if agonistic activity is desired, , Containing at least one O-glycosylation site. Some non-hormonal The lycosylated form is an antagonist and is thus useful. That CTP Uni The 121st (site 1), 127 (site 2), 132 (site 3), and 138 ( The serine residue at site 4) contains four such sites. For agonists of the present invention Useful CTP subforms are one or more of the ordered forms found in the native CTP sequence. It contains such a site. Therefore, the "moiety" used in the agonists of the present invention The CTP unit has four total glycosylation sites; sites 1, 2, and 3; site 1, 2, And 4; sites 1, 3, and 4; sites 2, 3, and 4; or simply site 1 And 2; 1 and 3; 1 and 4; 2 and 3; 2 and 4; or 3 and And 4 or may contain only one of sites 1, 2, 3 or 4. You.   "Vertical" inserts or extensions allow for fewer inserts or extensions Is also meant to contain two "CTP units". Each CTP unit is complete Alternatively, it may be a fragment, natural or variant. Vertical extension All CTP units in the insert or insert can be the same or different from each other. Can be Thus, for example, a vertically aligned extension or insert is typically a part- Complete; part-part; part-complete-part; complete-complete-part, etc., where Each of the partial or complete CTP units of interest independently contains a variant or It can be a native sequence.   The “linker moiety” is the same α as a member of the heterodimer, unless otherwise specified. Chain or β chain, or the activity Connects the α and β sequences without being hindered by the activity of converting to gonist activity. It is the part that Although the level of activity can vary within a reasonable range, the linker Impair the single-chain form of both substantial agonist and substantial antagonist activity It cannot exist in a way that makes it happen. Single-stranded form is single-stranded when recovered from production medium The dimers, ie, the forms of the elements that form the compound, are maintained in chain form. It must show a suitable activity relative to the mon activity.Mutant   Hormonal subunits and CTP units are either natural hormones or CTP sequences. It may correspond exactly or it may be a variant. The nature of the variant is defined above. Have been. In such variants, 1-10 of the amino acids contained in the native sequence, Preferably 1-8, and most preferably 1-5, compared to the natural amino acid at that position Replaced by different amino acids, or 1-10, more preferred 1-8, and most preferably 1-5 amino acids are simply deleted or Rui is a combination of these. As indicated above, the single-stranded form of non- Identified through detection of the presence of a critical region, or in particular a non-critical "loop" The number of amino acids modified by Can be increased to 20 or 30, or any number, depending on the length of the amino acid sequence of You. Of course, the effects can be affected by deletions or substitutions in more than one non-critical region. The substitution and deletion strategy yields more amino acids in the single-chain form affected Can be used in combination. Such substitutions or deletions may be cumulative, The agonist or antagonist activity is not substantially removed. Natural amino Substitution of the acid with a conservative analog is preferred.   "Conservative analog" is, in the conventional sense, the residue that is replaced is the residue that is replaced. It is an analog in the same general amino acid category as. Amino acids are As understood in the field, for example, Dayhoff, M. et al.Atlas of Protein Sequen ces and Structure (1972)Five: 89-99 classified into such groups . In general, acidic amino acids belong to one group and basic amino acids To one group, neutral hydrophilic amino acid to another group, and so on Belong.   More specifically, amino acid residues are generally classified into four major subclasses: Can be classified as:         Acidic: The residue has a negative charge due to the loss of H ions at physiological pH, Residues of the peptide are dependent on the aqueous solution when the peptide is present in an aqueous medium at physiological pH. To a surface position in the conformation of the peptide that is attracted and contains it Be sought out.         Basic: The residue has a positive charge due to its association with H ions at physiological pH , Its residue is dependent on the aqueous solution when the peptide is present in an aqueous medium at physiological pH. Surface position in the conformation of the peptide that is attracted by Is searched for in Oki.         Neutral / Non-polar: The residue is uncharged at physiological pH and the residue is When the peptide is present in an aqueous medium at physiological pH, it is repelled by an aqueous solution, It is sought at an internal position in the conformation of the peptide in which it is contained. These residues are also referred to herein as "hydrophobic".         Neutral / polar: The residue is uncharged at physiological pH and the residue is When the peptide is present in an aqueous medium at physiological pH, it is attracted by the aqueous solution. , Located in an outer position in the conformation of the peptide in which it is contained .   Of course, in a statistical collection of individual residue molecules, some molecules are charged. Some are uncharged and, to a greater or lesser extent, draw aqueous media. There is attachment or repulsion. To meet the definition of "charged", A significant percentage (at least about 25%) is charged at physiological pH. Polar The degree of attraction or repulsion required for classification as non-polar or non-polar is arbitrary. Thus, the amino acids specifically contemplated by the present invention have been classified as either. Especially Most unnamed amino acids can be classified based on known effects.   Amino acid residues may be cyclic or non-cyclic, and aromatic or non-aromatic , As a trivial classification of residue side-chain substituents, as well as Can be classified into subclasses. In addition to carboxylic carbon, total 4 or less carbon When containing an atom, the residue is considered small. Of course small residues , Non-aromatic.   For naturally occurring protein amino acids, subsequences according to the above scheme The Russ classification is as follows:         Acidic: Aspartic acid and glutamic acid;         Basic / Acyclic: Arginine, lysine;         Basic / cyclic: Histidine;         Neutral / Polar / Small: Glycine, serine, cysteine;         Neutral / Non-polar / Small: Alanine;         Neutral / polar / large / non-aromatic: Threonine, Asparagine, Glutamine ;         Neutral / Polar / Large / Aromatic: Tyrosine;         Neutral / Non-polar / Large / Non-aromatic: Valine, isoleucine, leucine, Thionin;         Neutral / Non-polar / Large / Aromatic: Phenylalanine, and tryptopha N.   The gene-encoded secondary amino acid proline is technically neutral / nonpolar / large / Within the cyclic and non-aromatic groups, but the secondary conformation of the peptide chain. This is a special case of known effects on Is included.   When the single chain proteins of the invention are constructed recombinantly, they are Containing only amino acid substitutions, but any of the moieties are standard methods, for example, Synthesized by solid phase peptide synthesis to the remaining protein, eg enzymatically , When linked, aminoisobutyric acid (Aib), phenylglycine (Phg) Non-gene-encoded amino acids such as may also be replaced with their similar binding pairs. You.   These non-coding amino acids can also be, for example, β-alanine (β-Ala), or Other amino acids such as 3-aminopropionic acid and 4-aminobutyric acid, Lucosin (Sar), Ornithine (Orn), Citrulline (Cit), t-Butylalanine (T-BuA), t-butylglycine (t-BuG), N-methylisoleucine (N-MeIle), And cyclohexylalanine (Cha), norleucine (Nle), cysteic acid ( Cya), 2-naphthylalanine (2-Nal); 1,2,3,4, -tetrahydroisoquinoline-3- Carboxylic acid (Tic); mercaptovaleric acid (Mvl); β-2-thienylalanine (T hi); and methionine sulfoxide (MSO). These are also convenient Applies to a particular category.   Based on the above definition,         Sar and β-Ala and Aib are neutral / non-polar / small;         t-BuA, t-BuG, N-MeIle, Nle, Mvl, and Cha are neutral / non-polar / large / Non-aromatic;         Orn is basic / acyclic;         Cya is acidic;         Cit, Acetyl Lys, and MSO are Neutral / Polar / Large / Non-Aromatic ; And         Phg, Nal, Thi, and Tic are neutral / non-polar / great / aromatic.   Various ω-amino acids, depending on size, are neutral / non-polar / small (β-Ala, ie , 3-aminopropionic acid, 4-aminobutyric acid) or large (all others) Is done.   Therefore, amino acid substitutions other than the gene-encoded amino acids also apply to the present invention. Peptide compounds within the scope can be included according to their general structure It can be classified as a chime.Preferred embodiment of single chain hormone   The single-chain hormone of the present invention is most efficiently and economically produced by the recombinant method. Produced. Thus, such forms of alpha and beta chains, CTP units, and Other linker moieties containing only gene-encoded amino acids are preferred. But on As described above, the single-chain form can be obtained by the synthetic peptide method or other organic synthesis method. It is possible to construct at least a part of the mon and therefore Variants containing deamino acids are also within the scope of the invention.   In the most preferred embodiment of the single-chain hormone of the present invention, the C-terminal of the β subunit The end is covalently attached to the N-terminus of the mature α subunit, optionally via a linker. Are connected by. C-terminal of α subunit was linked to N-terminal of β subunit The form is also useful, but it is an antagonist of the relevant receptor Alternatively, it may have somewhat lesser activity as an agonist. Concatenation of one subunit A direct peptide in which the C-terminal amino acid is directly linked to the other N-terminal via a peptide bond. It may be a peptide linkage, but in many instances a linker between the two ends. -Portion is preferred. In many cases, the linker moiety is less Provides at least one β-turn. Therefore, of the proline residue in the linker Presence can be advantageous.   As described above, in each case, the N-terminus of the α chain is linked to β via the linker unit. Can be coupled to the N-terminus of the chain, or the C-terminus of the α-chain is the C-terminus of the β-chain Can be coupled to two α subunits or two Included in single-stranded form containing the β subunit.   In considering linkage between subunit ends, including single-stranded forms, one or more ends It should be understood that the ends can be modified by substitutions or deletions as described above. I have come.   A preferred embodiment of a single chain compound containing two β subunits is one unit. The C-terminal of the unit via a linker (preferably a peptide linker), if necessary. And a compound linked to the other N-terminus. As mentioned above, the N-terminals of the two β chains End-to-end ligation or C-terminal ligation of two β-strands is also possible, of course, In this case a linker is required.   Head-to-head, tail-to-tail, and head-to-tail arrangements of both single-stranded heterodimers, and single Although the chain 2β subunit form has been described, the linkage between the two subunits is Proximity to N- or C-termini of a member, not exactly at that position It can also occur in position.   In one particularly preferred set of embodiments, the linkage is head-to-tail and the linker Part is one or more CTP units and / or their variants or truncations Including morphology. Preferred forms of CTP units used in such linker moieties Are listed below.   Furthermore, the linker moiety is preferably covalently linked, preferably releasably, to the linker. The drug may be attached to the moiety. A method of coupling a drug to a linker moiety, And the method of providing its release is conventional.   CTP and its variants and truncations, in addition to their presence in the linker moiety May also be contained in any non-critical region of the subunits that make up the single-chain hormone You. The nature and location of these inclusions are described in the parent application (pa rent application) is described in detail.   CTP units are the preferred inclusions in the linker moiety, but the linker is α Any suitable spatial relationship between the subunit and the β subunit It is understood that it can be a covalently bound substance. Therefore, in the head-to-tail configuration, the linker Is generally a suitable space and conformation in solution. Having a suitable hydrophilic / hydrophobic ratio to provide Is a peptide containing less than 100, more preferably less than 50 amino acids obtain. Generally, the linker is present in ambient solution to allow the α subunit and β It does not interfere with the interaction between the subunits or the two β subunits As it turns out, it is hydrophilic in the end. The linker is typically provided by a proline residue It is preferable that the β-turn is included. A peptide peptide that has the exact properties described above. Any suitable polymer can be used, including the linker.   One particular linker moiety not included within the scope of the present invention is a downstream subunit. Is a linker portion containing a signal peptide immediately upstream of the site.   Particularly preferred embodiments of the single chain hormone of the present invention include: The human form of the subunit is also particularly preferred. In the above construct, "CTP" is CTP or its variants or truncated forms, as further explained in the following section It is.Preferred embodiment of CTP unit   The symbols used for the CTP unit of the present invention are as follows: Complete CTP unit For the part of the part, the position included in the part is the number shown in FIG. 2 of the present application. Is shown. When a substitution is present, the substituted amino acid indicates that position. Shown with a superscript. So, for example, CTP (120-143) is 1 through 120 It represents the CTP portion extending to the 43rd position, and CTP (120-130; 136-143) is 118-119 of the native sequence. Represents fusion sequences lacking positions 131-135 and 144-145. CTP (Arg¹²²) Is , A variant in which the lysine at position 122 was replaced by arginine, CTP (Ile¹³⁴ ) Indicates a variant in which position 134 is replaced with isoleucine. CTP (Val¹²⁸Val¹⁴³) Has two substitutions, one is leucine at position 128 and the other is 142. Position isoleucine. CTP (120-143; Ile¹²⁸Ala¹³⁰) Is The relevant parts of the CTP unit with the two indicated substitutions are represented.   Furthermore, preferred CTP variants have one or more O-linked glycosylation sites. It has been modified or deleted. Mutate sites to prevent glycosylation A particularly preferred method is the replacement of serine residues at these sites with alanine residues. .   Particularly preferred are CTP units of the formula: Preferred embodiments of α and β subunits   Of course, the natural form of the α and β subunits in single-stranded form is preferred Within the embodiment. However, certain variants are also preferred.   In particular, the N-linked glycosylation site at position 52 is located at or near this site. Mutants of the α subunit that have been removed or modified by Preferred for tagonist activity. Similar modifications at the glycosylation site at position 78 are also preferred. New Deletion of one or more amino acids at positions 85-92 also results in a form containing the α subunit. Amino acid substitutions or deletions at these positions, which affect the nature of the mon activity, also This is a preferred embodiment.   Similarly, the N-linked glycosylation site in the β-chain appears to eliminate glycosylation. It can be modified in a favorable manner, and therefore has a β-chain agonist or antagonist activity. Can affect. CTP is present naturally as in CG or as a linker The O-linked glycosylation site of this moiety is also modified when present by Can be done.   Specific variants containing altered or deleted glycosylation sites are described by Yoo, J. et al.J Biol Chem (1993)268: 13034-13042; Yoo, J. et al.,J Biol Chem(1991)266: 177 41-17743; and Bielinska, M. et al.,J Cell Biol(1990)111: 330a (all above , And Matzuk, M.M. et al.,J Biol Chem(1989)264: 2409-2414; Keene, J .L. Et al.J Biol Chem(1989)264: 4769-4775; and Keene, J, L, et al.Mol Endocr inol (1989)Three: 2011-2017.   Not only can the glycosylation site itself be directly modified, but these The proximity of the sites also predominates so that the glycosylation status of the variant is affected. Be modified. For the α subunit, for example, the amino acids between the 50th position and the 60th position are located. Preferably (including both conservative and non-conservative substitutions), especially Asn₅₂so Because of their proximity to the glycosylation site of, positions 51, 53, and 55 Substitution is preferred.   Α subunit in which lysine at position 91 is converted to methionine or glutamic acid Mutants of are also preferred.   Variants have been discussed above for variations of individual subunits, It is recalled that the single-stranded form of the dimer offers an opportunity for modification. Especially the die Regions Important for Mer Folding Are Important for Accurate Conformation of Single-Stranded Molecules However, these regions are above-mentioned in terms of individual members in dimeric form. It allows for variations of single-stranded forms, although not described in. Furthermore, one The double-stranded form is such that alterations and / or deletions do not affect the biological activity as described above. It can be dramatically modified for non-critical areas.Suitable drug   Suitable agents that may be included in the linker moiety are insulin-like growth factor; epidermal growth factor. Offspring; acidic and basic fibroblast growth factor; platelet-derived growth factor; various colonies -Stimulatory factors (eg granulocyte CSF, macrophage CSF, etc.); Itokine (eg, IL-2, IL-3, and excess interleukintan Various interferons; peptides such as tumor necrosis factor Or protein. Single chain peptide or protein based drug The complete construct can be easily produced by recombinant expression of one gene. Has the advantage that In addition, like antibiotics, anti-inflammatory agents, toxins, etc. Small molecule drugs can be used.   Generally, the drug contained within the linker moiety is the receptor to which the hormone normally binds. It is a drug that is desired to act in the vicinity of the target. Since the drug is contained in the linker Suitable supplies to be released include, for example:Preparation methodMore on the section entitled By including a site for enzyme-catalyzed lysis, as described in Provided.Other modifications   The single-chain protein of the present invention has phosphorylation, glycosylation, and normal glycosylation. Deglycosylation of forms, modification of amino acid side chains (eg Conversion to lorin), and post-translational events generally accepted to occur It is generally understood to derivatize an amino acid sequence, such as similar modifications similar to It can be further complexed or derivatized in the manner described.   The glycosylation status of the hormones of the invention is of particular importance. Hormone is prokaryotic Any CTP that may be produced in the host, or may be subunits and / or present By mutating the glycosylation site normally present in the unit, non-glycosylation It can be prepared in silylated form. The non-glycosylated form of the hormone and partially Cosylated forms can be prepared by manipulating glycosylation sites . Usually, of course, glycosylated forms are also included within the scope of the invention. .   As is known in the art, the single chain proteins of the invention may also be tailored for the desired indication. It can then be coupled to labels, carriers, solid supports, etc. The sign form is It can be used to track their metabolic processes. Suitable labels for this purpose are , Radioisotope labels such as iodine-131, technetium-99 and indium-111. Include. The label also mediates the detection of single chain proteins in the assay system Can be used for In this case, the radioisotope also includes an enzyme label, a fluorescent label, It can be used in the same manner as a chromophore. The use of such labels makes them Child Being a receptor ligand, it is particularly useful for these proteins.   The proteins of the invention also specifically immunoreact with these novel modified forms. In preparing antibodies, the carrier may be coupled to enhance their immunogenicity. Can be A suitable carrier for this purpose is a keyhole limpet hemo Cyanine (KLH), bovine serum albumin (BSA) and diphtheria toxoid Includes. Carriers of modified peptides of the invention, including the use of bifunctional linkers Standard coupling methods for coupling to can be used.   Similar conjugation methods, along with other methods, can be used to bind proteins of the invention to solid supports Can be used to couple to. Once coupled, these tags The protein is then used to isolate the desired components for which a specific reaction is shown. It can be used as a reagent.Preparation method   Methods for constructing the proteins of the present invention are well known in the art. Above As shown in the table, only the gene-encoded amino acids are included, and the single strand has a head-to-tail configuration. Currently, the most practical approach is to generate DNA encoding the desired protein. Presently is to synthesize these substances by recombinant methods. One containing a variant DNA containing the nucleotide sequence encoding the double-stranded form was prepared from the native sequence. Can be Site-directed mutagenesis, ligation of additional sequences, PCR, and construction of appropriate expression systems. Methods for construction are well known in the art to date. Coat the desired protein Part or all of the DNA to be linked is preferably a restriction site for facilitating ligation. Can be constructed by standard solid phase methods. Coding arrangement included Appropriate control elements for sequence transcription and translation are provided for the DNA coding sequence. Can be As is well known, expression systems have a wide range of hosts (including prokaryotic hosts such as bacteria and Includes eukaryotic hosts such as yeast, plant cells, insect cells, mammalian cells, avian cells, etc. ) Is now available.   The choice of host is especially for post-translational events, most especially including glycosylation. It is. The location of glycosylation depends on the nature of the glycosylation site within the molecule. In most cases it is controlled, but the nature of the sugars that occupy this site is largely dependent on the host. Controlled by the nature of the Lord. Therefore, fine adjustment of the characteristics of the hormone of the present invention It can be achieved by the proper choice of the Lord.   A particularly preferred form of the gene for the alpha subunit part (alpha subunit is modified Or is not modified) is a "minigene" construct.   As used herein, the α subunit “minigene” is the pM²/ CG α or pM²In terms of the construction of / α, Matzuk, M.M. et al.Mol Endocrinol(1988)2 : 95-100 disclosed herein. This "minigene" is Characterized by retention of only the intron sequence between exon 3 and exon 4 And all upstream introns have been deleted. The specific constructs described The N-terminal coding sequence derived from Son 2 and a portion of exon 3 are supplied from cDNA. Via the XbaI restriction site, between exons I and II, and exons II and II. Directly in the coding sequence of exon 3 so that the intron between III and III does not exist. It is directly connected. However, the intron between exons III and IV, and the code The signal 3'of the swing sequence is retained. The resulting minigene is BamHI / BgIII It may be conveniently inserted as a segment. Other construction methods for suitable minigenes It is, of course, possible that the definition is that the coding sequence is linked via the XbaI site. You are not limited to any particular build. But this is a convenient means of gene construction Yes, for other approaches, such as synthetic or partial synthetic preparation of genes There is no particular advantage to it. Definition is an intron, between exons III and IV Or hold any other intron, preferably hold other intron No, including the coding sequence for the α subunit.   For recombinant production, modified host cells using the expression system are used and the desired Cultured to produce a protein. These terms are used herein as follows: Used for:   “Modified” recombinant host cells, ie, containing recombinant expression systems of the present invention. A cell that has been modified so that it can be introduced into Include this expression system by any convenient manner, including irs infection and the like. It is a modified host cell. “Modified” includes this expression system A cell, the system of which is either integrated into the chromosome or is extrachromosomal . A "modified" cell may be stable with respect to the inclusion of an expression system or may be stable. Fixed Cannot be. Briefly, a "modified" recombinant host cell containing the expression system of the invention is A cell is a cell that, as a result of the inclusion of this expression system when it is not naturally present, Cells regardless of the uptake mode of the expression system.   An "expression system" is the coding nucleotide sequence to be expressed and DN containing regulatory sequences with which they are required to affect expression of the coding sequences It is the A molecule. Typically, these controls are promoters, termination regulatory elements. Rows and, in some cases, operators or other that regulate expression Including the mechanism of. The control sequences are designed so that they are functional in the particular target recombinant host cell. And, thus, the host cell selected to suit the control sequences in the constructed expression system. It must be.   If secretion of the produced protein is desired, code for the signal peptide. The additional nucleotide sequence of the desired single-strand for producing the preprotein is also Included to produce a signal peptide operably linked to the hormone . Upon secretion, the signal peptide is truncated to release the mature single chain hormone. Refused.   As used herein, "cell", "cell culture" and "cell line" Are used interchangeably without paying particular attention to subtle differences in meaning. Those When the distinction between is important, it is clear from the context. If either is meant, then all Are intended to be included.   The protein produced can either be from a cell lysate when produced intracellularly, or Can be recovered from the medium when secreted. Recombinant protein from cell culture Methods for recovering protein are well known in the art and these proteins are Purified by known methods such as ruffy, gel electrophoresis, selective sedimentation, etc. obtain.   The whole or part of the hormone of the present invention can be synthesized by peptide synthesis methods known in the art. Therefore, it can be directly synthesized. The synthesized moieties can be linked and included in the linker moiety. Release sites for any of the agents may be introduced by standard chemical means. Heredity These embodiments containing amino acids that are not encoded by the offspring and head-to-head Of course, for those embodiments in which a ruby-tail-to-tail arrangement is used, The growth is at least partial at the protein level. Natural N-terminal or natural N-terminal The head-to-head connection at the position close to the edge is not compatible with amino groups such as dicarboxylic acid derivatives. It is provided via a linker containing the corresponding functional group. C-terminal or C-terminal Proximal tail-to-tail configurations are diamines, diols, or combinations thereof. Is provided via a linker.antibody   The protein of the present invention produces antibodies that specifically immunoreact with these novel compounds. Can be used to generate These antibodies are useful in various diagnostic and therapeutic applications It is. For example, the single chain form of the invention may be used therapeutically in humans or vertebrates. When tested, drug levels can be measured using conventional immunoassays for these antibodies. Can be monitored using. Furthermore, it was induced by these single-stranded forms Some antibodies cross-react with heterodimers, so Can be used to diagnose the levels present in.   Antibodies are standard in mammals such as rabbits, mice, sheep, or rats Commonly prepared by immunization protocols, and antibodies ensure sufficient immunity Is titrated as a polyclonal antiserum. Next, polycloner The antiserum is recovered, eg, for use in immunoassays. From the host Antibody-secreting cells of, for example, spleen cells or peripheral blood leukocytes can be prepared by known methods. Production of immortalized monoclonal antibodies immunospecific for the protein of the invention Can be screened for.   Determine affinity or non-affinity by "immunity specificity to protein" Within the general parameters considered to be An antibody that does not immunoreact with the telodimer itself is meant. Specificity is relative The term may be selected to be any limitation, such as a 100-fold or greater difference in immunoreactivity. It is understood that Accordingly, immunospecific antibodies encompassed within the invention are single chain At least 100 times more than the corresponding heterodimer for a protein It is reactive.Prescription   The proteins of the present invention are known for heterodimers corresponding to single chain forms. It may be formulated and administered by methods similar to certain methods. Therefore, the prescription and The method of administration will vary according to the particular hormone used. However, the dose level And the frequency of administration, especially for organisms in which the CTP unit is extended due to its presence. When present in view of the biological half-life, it can be altered compared to heterodimers. You.   The protein formulation of the present invention isRemington's Pharmaceutical Sciences,latest Proteins, as found in Edition, Mack Publishing Company, Easton, PA. Or it is a typical formulation for peptide drugs. In general, proteins are By intravenous, intramuscular, subcutaneous, or intraperitoneal injection, or transmucosal or It is administered using a formulation for transdermal delivery. These formulations are generally It contains an active agent or penetrant (for example, bile salt, fusidic acid, etc.). this These formulations are aerosols or suppositories, or, for transdermal administration, skin patches. It may be administered in the form of chi.   Oral administration is also possible if the formulation protects the peptides of the invention from degradation in the digestive system. It is also possible.   Dosage regimens and formulation optimizations should be performed in Runs as it is done.how to use   The single-chain peptides of the invention may be used in a variety of ways, most certainly the hormone It can be used as a substituent in the rhodimer form. Therefore, like the heterodimer , Agonistic forms of the single-chain hormones of the invention are found in both humans and animals. Related to infertility treatments, such as in vitro insemination aids, and natural hormones It can be used for other treatments.   Single-chain hormones are also useful as reagents in a manner similar to heterodimers. You.   Furthermore, the single-chain hormones of the present invention relate to native proteins in biological samples. Used as a diagnostic tool for detecting the presence or absence of the antibody. So They also provide an assay for assessing the levels of these hormones in various samples. It is useful as a control reagent in the kit. The level of the hormone itself, is Or protocols for assessing the level of antibodies elicited against them, Standard immunoassay protocol well known in the field. Different competition and direct A variety of assays are available, including radioisotope labels, fluorescent labels, enzyme labels, etc. It can be used with labeling methods.   The single-chain hormone of the present invention also detects the receptor bound by the natural hormone. And is useful for purification. Therefore, the single-chain hormone of the present invention is not bound to the solid support. Affinity chromatography of the receptor or anti-hormone antibody It can be used for rough preparation. The resulting receptors are screened for therapeutic and reagent candidates. The Leaning Test was used to assess hormone activity against a candidate drug. The body is useful.   Finally, the antibodies that react uniquely with the single-chain hormones of the present invention are unique to these substances. It can be used as a purification means for separation and subsequent preparation. They are also drugs Can be used to monitor the levels of single chain hormones administered as.   The following examples are intended to illustrate but not limit the invention. Absent.                                 Example 1                       Preparation of DNA encoding CGβ-α   Figure 1 shows the construction of inserts for expression vectors, where the C-terminus of the β chain of human CG. The end is linked to the N-terminus of the mature human α subunit.   As shown in FIG. 1, the polymerase chain reaction (PCR) is a method for exocytosis of CGβ. To fuse the two subunits between protein 3 and exon 2 of the α subunit , The carboxy terminal amino acid codon of CGβ is the N-terminal of the α subunit. It is fused directly to the codon for the terminal amino acid in open reading frame. This is a hybrid Using a Limer to amplify a fragment containing exon 3 of CGβ More complete, the hybrid primer contains the N-terminal sequence of the α subunit. It has a coding "tail". Therefore, the obtained amplified fragment contains human CGα It contains the portion of exon 2 that encodes.   Independently, the N-terminus of the α subunit fused to the codon corresponding to the C-terminus of CGβ Hybrid primers encoding end sequences are used to amplify the α minigene. Used as one of the primers. Each codes the other subunit The two amplified fragments containing the overlapping portion of Amplified together with two additional primers that cover the full sequence stretch.   More specifically, reaction I is associated with exon 3 of CGβ and the maximum of the mature α subunit. A fragment containing the first 4 amino acids and a SalI site in the 5'-direction of the coding sequence. FIG. Matzuk, M.M. et al.Proc Natl Acad Sci USA(19 87)84: 6354-6358; Policastro, P. et al.,J Biol Chem(1983)258: 11492-11499 on Obtained by amplifying a portion of the described CGβ genomic sequence.   Primer 1 provides a SalI site and has the following sequence:   The other primer, primer 2, contains 4 codons of the αN-terminal sequence and It is complementary to the 5 codons of the C-terminal sequence of CGβ and has the following sequence:   Therefore, the resulting amplified segment, which is the product of reaction I, contains the fusion coding region. It has a SalI site in the 5'-direction of the region.   In reaction II, a similar fused coding region from the α minigene described above can get. Primer 3 contains 4 codons for the β subunit and 5 codons for α. Is a hybrid primer and has the following sequence:   Primer 4 contains a SalI site and is complementary to the extension of α exon 4. Step Limer 4 has the following sequence:   Therefore, the products of reactions I and II are overlapping, and primer 1 and The desired SalI product as shown in reaction III when subjected to PCR in the presence of 4. Cause   An amplified fragment containing the CGβ exon 3 and α minigene was prepared by Sachais,  B., Snider, R.M., Lowe, J., Krause, J.J Biol Chem(1993)268: 2319 PM containing CGβ exons 1 and 2 in the manner listed²With the derived expression vector PM²It is inserted into the SalI site of HA-CGβ exons 1 and 2. CGβ exon 1 and PM containing 2²Matzuk, M.M. et al.Proc Natl Acad USA(1987)84: 6354-6358, And Matzuk, M.M., et al.J Cell Biol(1988)106: 1049-1059.   This expression vector then produces a single-chain form of human CG, where the β-subunit The C-terminus of the knit is directly linked to the N-terminus of the α subunit.                                 Example 2                       Production and activity of single-chain human CG   The expression vector constructed in Example 1 was added to the Chinese hamster ovary (CHO). Cells were transfected and protein production was radiolabeled on SDS gels. It was evaluated by immunoprecipitation of the protein. The culture medium is collected and the single chain protein Bioactivity was compared to heterodimer in a competitive binding assay for human LH receptor. Compared. In this assay, cDNA encoding all human LH receptors was expressed in expression vectors. Inserted into pCMX (Oikawa, J.X-C et al.Mol Endocrinol(1991)Five: 759-768) . 293 exponentially growing cells were transferred to Chen. C. et al.Mol Cell Biol(1987)7: 2745-2752 Was transfected with this vector by the method described in.   In the assay, cells expressing the human LH receptor (2 x 10^FivePieces / tube) , 18 hours at 22 ° C with 1 ng of labeled hCG in competition with the sample to be tested. I had a cube. The sample was then placed in cold Dulbecco's PBS (2 ml) supplemented with 0.1% BSA. ), And the mixture was centrifuged at 800 × g for 15 minutes. Pellet with Dulbecco's PBS After washing twice, radioactivity was measured with a gamma counter. Specific binding is In the absence, 10-12% of all labeled (iodinated) hCG added. Was. The reduction of label in the presence of sample measures the binding capacity of the sample. This assay So, for the human LH receptor in 293 cells, wild-type hCG showed 0.47 ng of ED₅₀Have Then Single-chain protein is 1.1 ng ED₅₀It had.   Further assays for agonist activity evaluated stimulation of cAMP production . In this case, 293 cells expressing the human LH receptor (2 x 10^Five(Piece / tube) Incubated with varying concentrations of heterodimeric hCG or single-chain hCG And cultured for 18 hours. Extracellular cAMP levels were measured by Davoren, J.B. et al.Biol Reprod(19 85)33: 37-52, by specific radioimmunoassay. In this assay, the wild type had 0.6 ng / ml ED₅₀And the single-chain form has 1.7 ng / ml It had an ED50. (ED₅₀Is 50% of the effective dose. )   Therefore, in all cases, the effects of both wild-type and single-chain forms are similar. You.                                 Example 3                           Further activity assay   CH transfected with expression vector for βFSH-CTP-α single chain construct Media from O cells was harvested and evaluated as described in Example 2. To FSH receptor The results of the competition assay for the binding of E. coli are shown in FIG. The result is that the single-stranded form is , In the inhibition of FSH binding to its receptor, CTP extension to wild type FSH or β chain It is shown to be more effective than any of the FSH, including the length. E of the extension heterodimer D₅₀Is slightly above 100 mIU / ml, but the single-chain form of ED₅₀Is about 50 mIU / ml. Wild type FSH ED₅₀Is about 120 mIU / ml.   The results of the signal transduction assay are shown in Figure 4. Effectiveness of all three forms of FSH Are equivalent.                                 Example 4                        Construction of additional expression vectors   In a manner similar to that described in Example 1, single chain FSH, TSH, and LH (βFSH-α, βFS H-CTP-α, βTSH-α, β-TSH-CTP-α, βLH-α, βLH-CTP-α) Was prepared and transfected into CHO cells. The obtained hormone is Similar activity to the wild-type form when assayed as described in Example 2. Shows activity.   Similarly, a "2-β" single chain form of the expression vector is described in Example 1. Was constructed, expressed and assayed as described in Example 2 in a manner similar to that of.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI A61K 38/27 9051-4C A61K 37/38 C07K 14/59 9051-4C 37/02 16/26 9051-4C 37 / 36 C12N 5/10 9051-4C 37/43 C12P 21/02 9051-4C 37/66 // (C12P 21/02 C12R 1:91) (31) Priority claim number 08 / 351,591 (32) Priority Date December 7, 1994 (33) Priority claiming country United States (US) (81) Designated country EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, M C, NL, PT, SE), AU, BR, CA, CN, FI, HU, JP, KR, MX, NO, RU

Claims (1)

[Claims]   1. Glycosylated or non-glycosylated protein, said protein Is   Amino acid sequence of one β subunit of glycoprotein hormone, if necessary Covalently linked via a linker moiety to the glycoprotein hormone. Contains the amino acid sequence of the common α subunit,   Here, the α and β subunits are the natural amino acid sequence or the amino acid Consisting of sequence variants, protein.   2. The protein comprises the linker moiety, and the linker moiety is If necessary, drugs that should be targeted to the glycoprotein hormone receptor The protein according to claim 1, which is contained.   3. The position close to the C-terminal of the β subunit may be mediated by a linker moiety if necessary. And is covalently linked to the N-terminal proximal position of the α subunit, or Means that the position close to the C-terminal of the α subunit is optionally linked via a linker moiety. And being covalently linked to the N-terminal proximal position of the β subunit. The described protein.   4. The β subunit is a human chorionic gonadotropin or a variant thereof. Β subunit of   Whether the β subunit is the β subunit of FSH or a variant thereof, Or   The β subunit is a complete or partial CTP unit or a variant thereof. Is a β subunit of FSH extended near its C-terminal, or   Whether the β subunit is the β subunit of LH or its variant, Ruiha   The β subunit is a full or partial CTP unit and its C-terminal Is the β subunit of LH extended near the edge, or   Whether the β subunit is a β subunit of TSH or a variant thereof, Or   The β subunit is a complete or partial CTP unit or a variant thereof. The β subunit of TSH extended near its C-terminus and / or Is   The α subunit is a complete or partial CTP unit or a variant thereof. Extended to a position near its N-terminal, The protein according to claim 1.   5. The α subunit or β subunit or both are complete or Insert partial CTP units or variants thereof into their non-critical regions Modified and / or the linker moiety is complete or partial. The protein according to claim 1, comprising a CTP unit or a variant thereof.   6. The partial CTP unit is 112-132, 115-132, 116-132, or 11 From 8-132, 112-127, 115-127, 116-127, or 118-127 And / or   The CTP is modified or deleted with one or more O-linked glycosyls And / or   The non-critical region is adjacent to the C-terminus, or   The non-critical region is adjacent to the N-terminus, The protein according to claim 5.   7. The β subunit is one or more N-linked glycosylation moieties Position modification, and / or   One or both of the N-linked glycosylation sites of the α subunit have been modified. And / or   It is not glycosylated and / or   One or more amino acids at positions 85-92 of the α subunit have been deleted Was The protein according to claim 1.   8. Glycosylated or non-glycosylated protein,   Amino acid sequences of β subunits of the same or different glycoprotein hormones Glycoprotein, which is covalently linked to the protein via a linker moiety as necessary. The amino acid sequence of the β subunit of the hormone,   Here, the β subunit is the natural amino acid sequence of the hormone, or An amino acid sequence consisting of a variant of the amino acid sequence; or   Amino acid sequence of the α subunit of the same or different glycoprotein hormones Glycoprotein, which is covalently linked to the protein via a linker moiety as necessary. The amino acid sequence of the α subunit of the hormone,   Here, the α subunit is the natural amino acid sequence of the hormone, or An amino acid sequence consisting of a variant of the amino acid sequence; , Including proteins.   9. C-terminal proximity of one α or β subunit is in the other α Or at a position near the N-terminal of the β subunit, optionally via a linker moiety, The protein according to claim 8, which is covalently linked.   10. 9. The protein of claim 8, wherein the protein comprises a linker moiety.   11. Protein according to claim 1 or 8 in admixture with a suitable pharmaceutical excipient. A pharmaceutical or veterinary composition containing a quality.   12. An antibody that is immunospecific for the protein of claim 1 or 8. .   13. A nucleotide sequence encoding the protein according to claim 1 or 8. DNA or RNA molecule containing.   14． An expression system for producing a single-chain form of a glycoprotein hormone, said expression system comprising: The system comprises a first nucleotide sequence encoding the protein of claim 1 or 8 Included operably linked to a control sequence capable of affecting the expression of said first nucleotide sequence. Mu, expression system.   15. The protein encoded by the first nucleotide sequence is operable. A second nucleotide sequence encoding an operably linked signal peptide. 15. The expression system of claim 14 having.   16. A host cell modified to include the expression system of claim 15.   17． A method for producing a single chain form of a glycoprotein hormone, comprising:   17. The method of claim 16, wherein the method is under conditions where the glycoprotein hormone is produced. Culturing the cells of, and   Recovering the glycoprotein from the culture A method comprising: