JPH02207099A - Peptide relating to pthrp, preparation and use thereof - Google Patents

Abstract

NEW MATERIAL:A polypeptide represented by formula, etc., not having any human parathyroid hormone-relating protein(PTHrP) activity and having an antagonistic activity against the physiological action of the human PTHrP or a peptide having the human PTHrP activity. USE:A calcium metabolish remedy or a hypercalciuria remedy. PREPARATION:For example, a PTHrP gene is divided with a restriction enzyme into PTHrP(A) and PTHrP(B), which are synthesized into DNA fragments by a phosphoamidite method, respectively, and subsequently converted into genes corresponding to partial peptides by a ligation reaction. The genes are inserted into cloning vectors, and Escherichia coli transformed with the vectors is cultured. Plasmids are extracted from the train to give pCU-PTHrP(A) and (B), which are treated with a restriction enzyme and combined with expression vectors. Escherichia coli is transformed with the treated expression vectors and the transformed strain is cultured, followed by providing a polypeptide from the cultured product.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel PTHrP-related peptide, its production method, and its use as a therapeutic drug for calcium metabolism. The antagonists of this invention are used in the treatment of hypercalcemia.

[Prior Art] Parathyroid hormone is a typical regulator of calcium metabolism in the blood.
One, PTHI, calcitonin, vitamin D, etc., but it has been pointed out that none of the above is a causative agent of hypercalcemia caused by cancer.

In 1987, Mo5eley et al. isolated a protein exhibiting the same activity as PTH from a human squamous cell carcinoma exhibiting hypercalcemia.
related Protein, PTHrP
) was named. It has also been found that PTHrP is the causative agent of hypercalcemia in cancer patients.

Furthermore, the amino acid sequence and cDNA base sequence of PTHrP were determined by 5uva et al.
It was found that rP consists of 141 amino acids. 5uva et al. expressed PTHrP in a complement cell system using the obtained cDNA, and produced 48 μg of PTH activity.
/l expression has been confirmed.

Rodan et al. have already identified 1-34t of human PTHrP! The eye peptide fragment was chemically synthesized and used in the rat osteosarcoma-derived cell line ROSl? Human PTH fl-34 for increased activity of azonylate cyclase at /2.8
It has been confirmed that it has the same activity as 1.

Hypercalcemia in cancer patients accounts for about 1% of all cancer patients.
It is said to occur in 0% of patients, and the rise in blood calcium concentration causes various disorders such as pain.

Currently, calcitonin-related peptides are available as therapeutic agents for hypercalcemia, but their effects are temporary and the efficacy rate is low because calcitonin binds to receptors on osteoclasts.

Although it exhibits an inhibitory effect on bone resorption, this is because it does not suppress the activity of PTI (rP), which is a causative agent of hypercalcemia, and therefore its effectiveness is limited.

[Problems to be Solved by the Invention] Therefore, the object of the present invention is to provide a novel PTHrP that can act as an antagonist to PTHrP and treat hypercalcemia caused by PTHrP.
T) To provide an IrP-related peptide and a method for producing the same.

[Means for Solving the Problems J] As a result of intensive research, the inventors of the present application found that although human PTHrP does not have human PTHrP activity, it binds to receptors on bone cells to which human PTHrP binds, thereby increasing human PTHrP activity. We succeeded in obtaining a new polypeptide with antagonistic activities and completed this invention.

That is, the present invention provides a polypeptide having an activity that antagonizes the physiological effects of human PTI (human PT having rP activity) lrP or a polypeptide having human PTI lrP activity.

The present invention also provides a therapeutic drug for calcium metabolism against human PTHrP, which contains the above-mentioned polypeptide as an active ingredient.

Furthermore, the present invention involves transforming E. coli with an expression vector that includes a region encoding the polypeptide of the present invention and is capable of expressing the polypeptide in E. coli, and transforming E. coli with the transformed E. coli! Provided is a method for producing the polypeptide of the present invention, which comprises culturing a bacterium and recovering the polypeptide from the culture.

[Effects of the Invention] The present invention provides a novel polypeptide that is highly effective in suppressing hypercalcemia caused by human PTt (rP) in patients with PTH. There is [Tyr”I
b-PTH (significantly stronger than 7-341 (human PT
)l activity.

Furthermore, according to the production method of the present invention, it is possible to express a polypeptide in a large amount 500 times or more compared to the conventional system using mammalian cells by 5uva et al.

[Detailed Description of the Invention] As mentioned above, one polypeptide of the present invention comprises human PT)
Human PTI (rP or human PTHr) having rP activity
As a result of intensive research, the present inventors have discovered that human P
THrP is the 1st to 6th amino acid position counting from the N-terminus (hereinafter, unless otherwise specified, the amino acid position is N
Ala V, which is the amino acid sequence of
When al Ser Glu His Gin is missing,
Losing its PTHrP activity.

and L, which is the 7th to 34th amino acid sequence.
eu 1. eu old s Asp Lys Gly Lys
Ser Ile GlnAsp Leu Arg A
rg Arg Phe Phe Leu His Hi
s Leulle Ala Glu lie His
It has been found that the presence of Thr Ala can inhibit the physiological activity of human PTHrP by binding to receptors on bone cells to which human PTHrP binds.

Therefore, in one preferred embodiment of the present invention.

The polypeptide of the present invention does not contain the 1st to 6th amino acid sequences of human PTHrP, but does contain the 7th to 34th amino acid sequences. This polypeptide may contain an additional amino acid sequence, for example, the amino acid sequence after the 35th amino acid sequence of human PTHrP. In order to promote the expression of
It may have various E. coli-derived proteins or polypeptides attached to its terminal, but human PTH
A polypeptide having the 76th to 34th amino acid sequence of rP has activity as an anti-PTHrP antagonist, and therefore may consist only of this amino acid sequence.

In general, the physiological activity of polypeptides is as follows.

It is well recognized by those skilled in the art that even if a small number of amino acids constituting the polypeptide are substituted, deleted, or added, the polypeptide may be maintained. Therefore, some amino acids in the 7th to 34th amino acid sequences of the above human PTHrP are substituted, deleted, or a small number of amino acids are added thereto, and they do not have human PTHrP activity. , polypeptides having anti-human PTHrP antagonist activity are also included in the polypeptides of the present invention, and are particularly interpreted as having the amino acid sequence set forth in claim 22. In particular, those in which the 3rd to 15th amino acid sequences of human PTHrP are deleted and those in which the 2nd to 15th amino acid sequences counting from the C-terminus
It has been confirmed that a polypeptide in which amino acids on the C-terminal side of the 5th amino acid are sequentially deleted can achieve the object of the present invention. Furthermore, the polypeptides of the present invention also include polypeptides having anti-human PT) IrP antagonist activity, in which amino acids are substituted with non-naturally modified amino acids and which have human PT) IrP activity.

The drug for treating calcium metabolism of the present invention contains the polypeptide of the present invention as an active ingredient.

The dose of the therapeutic agent for calcium metabolism of the present invention for humans is usually about 3 x 10-8 mol to 3 x 10-' mol of the polypeptide of the present invention, and the preferred route of administration is intravenous or intramuscular injection. ,Also.

As a specific example of the formulation of an antagonist, a polypeptide of the invention may be prepared in physiological saline or citrate buffer.
10-'M to l x 10-'M can be mentioned.

The therapeutic agent for calcium metabolism of the present invention can be used to treat various diseases in which calcium metabolism is abnormal, such as hypercalcemia, bone diseases such as osteoporosis, and hypercalcemia due to chronic renal failure. .

The polypeptide of the present invention can be produced by chemical synthesis or genetic engineering techniques. When the number of amino acids is 40 or less, chemical synthesis is convenient; however, when the number of amino acids exceeds 40, chemical synthesis becomes difficult, so it is preferable to synthesize using genetic engineering techniques.

Methods for chemically synthesizing polypeptides are well known in the art and can be carried out using commercially available peptide synthesizers, such as the Abride Biosystems Model 43.
F, using a 0 peptide synthesizer. . It can be done by law.

Synthesis of the polypeptide of the present invention by genetic engineering techniques involves transforming Escherichia coli with an expression vector that includes a region encoding the polypeptide and is capable of expressing the polypeptide in Escherichia coli. cultivated,
This can be carried out by collecting the above-mentioned polypeptide from the culture.

In the above expression vector, 1) the region encoding the polypeptide of the present invention may have any base sequence as long as it encodes the polypeptide of the present invention; The following sequence is preferable as a nucleotide sequence encoding the 7th to 34th amino acids of human PTHrP, in which it is desirable to use codons with a high degree of codon and avoid sequences such as valindrome.

CTG CTG CACGACAAA GGT AAA
TCT ATCCAA GATCTG CGT CG
CCGT TTCTTCCTG CACCACCTG
ATCGCT GAA ATCCACACT GCA Upstream of the polypeptide coding region of the present invention, there is a promoter that increases transcription efficiency in E. coli. The promoter is preferably derived from E. coli, and the tryptophan promoter is particularly preferred. The polypeptide coding region of the present invention may be located immediately downstream of the promoter, but as shown in the Examples below, In the latter case, the polypeptide of the present invention can be obtained in the form of a fusion protein.

The expression vectors of this invention, like conventional expression vectors, have appropriate selection markers such as antibiotic resistance and an origin of replication for replication in E. coli. Furthermore, a transcription termination codon exists downstream of the polypeptide coding region of the present invention. These include, for example, pUc9, pBR32
2.Other commercially available vectors for E. coli can be used as they are.

The above expression vector is obtained by synthesizing the above-described polypeptide coding region of the present invention by a known method such as the phosphoramidide method, and cloning this into a commercially available vector for E. coli or a known expression vector that is expressed in E. coli. It can be produced by In the examples described below,
pAT-TrpET, which has an E. coli tryptophan promoter and expresses TrpE and TGF-a in E. coli
A partial PTHrP coding region was cloned into GF a (described in Japanese Patent Application No. 63-28908).

Transformation using the above expression vector can be carried out in exactly the same manner as transformation using conventional E. coli vectors. Furthermore, the culture conditions for E. coli can be the same as conventional ones.

The polypeptide of the present invention produced by E. coli transformed with the above vector can be obtained by collecting bacterial cells by centrifugation, destroying the bacterial cells by well-known norizozyme treatment and/or ultrasonication, and then gel-filtering the cells. 1. Specific conditions for separation and purification, which can be carried out by subjecting to chromatography or the like, will be described in detail in Examples below.

[Examples] Hereinafter, this invention will be described in more detail based on Examples, but this invention is not limited to the following Examples.

In addition, in the following examples, each operation is performed by T, Maniatis, "Mo1ec" unless otherwise specified.
ular Cloning, A Laborator
y Llanuaji (19821, Col. Spri
This was carried out by the method described in ng+1arbor.

Construction of Cloning Vector As shown in Figure 2.3, the PTHrP gene was cloned into (A) and (
B), and DNA fragments each consisting of about 60 bases were synthesized by the phosphoramidite method. The synthesized fragments were purified by reverse phase chromatography, and a ligation reaction was performed on the first half (A) as shown in Figure 4. .

A gene corresponding to the partial peptide (A) consisting of approximately 200 base pairs was obtained. Furthermore, the same operation was performed for the latter half (B).

The 5° and 3° ends of the obtained genes (A) and (B) both have the restriction enzyme Eco81 site and Sal1 site, and the cloning vector digested with EcoRI and Sal [cloning vector +) [inserted into IC9] (Figure 5),
This was used to transform Escherichia coli JM107 strain, and 40u
g/ml ampicillin, I PTG and X-gal
Candidate strains were obtained by culturing overnight in the presence of L medium.

After extracting the plasmid from the obtained candidate strain, the base sequence of the inserted gene was examined using the Sanger method, and it was confirmed that the gene sequence was as designed. PTH for this purpose
Strains with cloning vectors containing rP(Al and PT)lrP(81) were transformed into pUc-PTHrP, respectively.
(^) and pUc-PTllrP (named 81).

(2) Construction of direct expression vectors for partial peptides (A) and (B).

The construction of a direct expression vector for the first partial peptide (A) will be described below.

The pUc-PTI(rP (A) gene fragment was extracted and ligated with the large fragment of the expression vector pAT-X, which had been separately digested with the restriction enzyme C1a1.5aLI, using T4 ligase. Using this, E. coli strain 118101 was transformed. L in the presence of 40 gg/ral ampicillin.
A candidate method was obtained by culturing overnight in a medium. A plasmid was extracted using the obtained candidate method, a strain containing the desired inserted gene was obtained using restriction enzymes, and this was transformed into pAr-PTHrP I
The AI/HB strain was 101.

The same applies to partial peptide (B).

The obtained strain was transformed into pAT-PTHrP(Bl/)18101
It was made into a stock.

(3) Construction of an expression vector as a fusion protein of partial peptides (A) and (B).

The 1-part peptide (A) will be described below.

pUc-PTIIrP IAI/JL4107 strain with restriction enzyme EcoR[.

PTHrP (A
Extract the t gene fragment and use the restriction enzyme EcoRI,
The expression vector pAT-TrpE (G
The F-a large fragment was ligated using T4 ligase. E. coli strain 11BIOI was transformed using this and cultured overnight in L medium in the presence of 40 g/a+1 ampicillin to obtain a candidate method. A plasmid was extracted using the obtained candidate method, a strain containing the desired inserted gene was obtained using restriction enzymes, and this was transformed into pAT-TrpE-PTHrP.
(It was designated as 81/HBIOI strain.

141 Construction of direct expression vector of PTHrP pUc-
PTHrP IAI/J old 07 strain and pUc-PTIl
rP (Bl/JM107 strain each with restriction enzyme C1a
1. KpnI and) [pnl, cut with 5alI,
The first half gene of PTllrP of about 200 base pairs and about 2
6 and the expression vector pAT-X digested with the restriction enzyme C1a1.5alI to obtain a 10 base pair second half gene.
The large fragment of was ligated using T4 ligase (
Fig. 6) was used to transform Escherichia coli 1 (8101 strain) and cultured overnight in L medium in the presence of 40 ug/+1 ampicillin to obtain a candidate method. Plasmids were extracted from the obtained candidate method. As a result of restriction enzyme analysis, PT)Ir
The insertion of the Pifi gene was carried out by U&M, and this strain was transformed into pAT-
It was designated as PTHrP/11B101 strain.

151 Construction of expression vector for PTIIrP fusion Kumbaku protein pUc-PTtlrP IAI /JM107 strain and U
pUc-PTHrP (Bl/Jl1107 strain, restriction enzymes EcoRI, KpnI and Kpnl,
5all to obtain a PTHrP first half gene of about 200 base pairs and a second half gene of about 210 base pairs.
Both and the large fragment of the expression vector pAT-TrpE-TGF-a digested with the restriction enzyme C1a1.5alI were ligated using TR ligase. E. coli strain 118101 was transformed using this and cultured overnight in L medium in the presence of 40 gg/ml ampicillin to obtain a candidate method.

A plasmid was extracted using the obtained candidate method, and as a result of restriction enzyme analysis, insertion of the PT)IrP gene was confirmed, and this strain was transformed into pAT-TrpE-PTHrP/HBIO1.
It was made into a stock.

(6) Expression of various vectors The above pAT-PTHrP (
Al/) 18101 strain, pAT-pTHrP (81/
118101 strain, pAT-TrpE-PTHrP (
^)/H101 strain, pAT-TrpE-PTHrP
(Bl/)111101 strain, pAT-PTHrP (
Al/HB l 01 strain or pAT-TrpE-PT
The llrP (Al/118101 strain) was cultured under the same conditions as above to examine the expression of the recombinant protein.

Hereinafter, pAT-TrpE-PTI(rP(Al/
II old and old stocks will be described, but the treatment for other stocks is substantially the same.

pAT-TrpE-PTHrP fAl /H old and old strains were added to 32 ml of L containing 40 ug/ml ampicillin.
After overnight culture in the medium, 3.212 cells were inoculated into M9 medium containing 0.5% casamino acids and incubated at 37°C.
After culturing until the absorbance at 0 nm was 0.5, indole acrylic acid was added to a final concentration of 30 n+g/
After adding the cells to a concentration of I2 and continuing culturing for an additional 20 hours, 6 g of bacterial cells were collected by centrifugation.

The collected bacterial cells were treated with 2 ng/ml lysozyme, 2a+ME
DTA and U 100 mM) 'J Su-t'A acid m1
tl solution (pH 8, in a solution containing ol'!! cloudy, 0
℃ for 30 minutes. Furthermore, 35 μl of β-mercaptoethanol was added.

The bacterial cells were crushed by ultrasonication. - After stirring overnight, the soluble fraction (supernatant) and the insoluble fraction (precipitate) were separated by centrifugation, and each was subjected to 15% 5O8-polyacrylamide gel electrophoresis in the presence of 8M urea according to the method of 5tank and Munres. The desired PTHrP was added to the soluble and insoluble fractions at a ratio of 9:1, respectively.
(^) Protein expression was confirmed. Also, PTH
When the expression was investigated using a polyclonal antibody against a synthetic peptide of rP with a length of 1 to 34 amino acids, 30 mg7
The expression level of PTHrP (At) of 42 or more was obtained as the immunological activity. The obtained soluble fraction was freeze-concentrated, and separated and purified by Sephadex G-75 (manufactured by Pharmacia) gel filtration column chromatography. The specific conditions for gel filtration are eluent: 20+M Tris-HCI
(pH 8.01, column: φ2. x 100 am
, flow rate 2 m 1715 minutes.

The eluted fractions were collected and frozen, and the TrpE portion was removed and cleaved with cyanogen bromide. This was done by adding PTHrP to 70% formic acid so that the concentration of kungbaku substance was 1%, adding 100 equivalents of cyanogen bromide, and leaving the mixture at 37° C. for 24 hours. Furthermore, Sephadex G-
Gel filtration column chromatography was performed using 50 (manufactured by Pharmacia). The specific conditions for this gel filtration are:
20 mM TrisHCI (pH 8.01, column: medium 2°6 x 100 am, flow rate 2.2 all
/15 minutes. The eluate fractions were collected and freeze-dried to obtain 20 mg of PTHrP (Al kumbaku substance. The purity of this substance was determined by reverse phase column chromatography and 5D
It was confirmed by S-polyacrylamide gel electrophoresis that it was a single product.

F, using a Abride Biosystems Model 430 Peptide Synthesizer. . Human PTHr by method
P17-341NHx was synthesized.

The in vitro and in vivo effects of the synthesized human PTHrP (7-341NH*) were tested.

1Nvitro method: Rat osteosarcoma-derived cell line ROSl?/2.8 (Consigned number Sacho, +Acta Endocrinology)
ca-, vol, 116. p.

113, f19871 in Ham's F12 medium
Cultured for 1 day. F12 medium (Tris 10 mM,
BSA 0.1%.

1 [IMX 0.5 mlJ? - After cleaning the ivy,
Human PTHrP f7-341 NL fragment and human PTHrP fl-341NH* at different concentrations were added and incubated at 37°C for 10 minutes. On ice, IN
After adding HCI, the cells were collected, and the concentration of the supernatant was determined using a commercially available (Yamasa) CAMP RIA kit.

As a result, the antagonism of human PTHrP f7-341NIIz against human PT+lrP (1-341NH,
Tyr”]bPTHf7-34).

Figure 8 shows that PTI (rP (7-3
This is a diagram examining the inhibitory effects of various PTHrP synthetic fragments when the adenylate cyclase activity on RO31772.8 cells of 41 was set at 100.
7-341 MHz was inhibited most strongly, indicating that it is an excellent antagonist.

Furthermore, Figure 9 shows that the concentration of PTHrP (1-341) can be increased, and from this result, PTI
It was demonstrated that IrP f7-341 NHZ indeed acts as an antagonist.

flO51772,8 was cultured in the same manner as above, and HBS
After washing with S, a certain amount of ['''' I ] −r
PT Hr P (1-341N II z and unlabeled human PTIIrP (7-
341NHt fragment was added and incubated for 2 hours at room temperature. Elute with IN NaOH.

112′ PT bound to receptor by γ counter
HrP (1-341NH*a1 degree was measured. As a result, human PTHrP (7-341NHs is a conventional antagonist [Tyr”]]bPT former 7-34Nl
It was found that the receptor binding ability was superior compared to lt (Fig.
I tumor culture strain Lu61 (lung squamous epithelium P,) and Fujo
Tests were conducted using a model experimental system using nude mice with hypercalcemia and explantation of pancreatic cancer (pancreatic cancer).

Human PTllrP (0.1% 7-341Nil!
[ISA-containing citrate buffer (pH 6, dissolved in 511 ml) was administered through the tail vein of nude mice to give an IQ mg/mouse.The administration period was continuous administration for Lu61 cells 3 hours and 5 hours after administration. ,Fujok
For a cells, only the first one was used.

Blood was collected immediately after and at the points shown in FIGS. 11 and 12, respectively, and serum calcium concentration was measured by atomic absorption.

As a result, human PTHrP (7-341NHt) had no effect on normal mice, and only in mice that were transplanted with Lu61.Fujoka and developed hypercalcemia, calcium concentration decreased to normal levels over a long period of over 36 hours. (FIG. 11 and FIG. 12), 10 ug/mouse was equivalent to 500 μg/human, which was an amount that would pose no problem as an actual therapeutic agent.

As described above, the therapeutic agent of the present invention has a sustained effect of several tens of hours or more after one administration, and B! <Hado is excellent,
It is useful as a therapeutic agent for bone diseases and diseases involving the central nervous system or as a clinical diagnostic agent.

[Brief explanation of the drawing]

Figure 1 shows PTllrP in the expression vector of this invention.
Restriction enzyme maps near the coding region, Figures 2 and 3 are diagrams showing the base sequences of genes encoding PT)IrP(At and PTHrP (81), respectively. Figures 4 to 7 show the construction of the expression vector of the present invention. Diagram for explaining the operation. Figure 8 is a diagram showing the inhibitory effects of various PTHrP synthetic fragments on ROS17/2.8 cells of PTHrP(7-34) having PTHrP activity when the adenylate cyclase activity is set to 100. Figure 9 is a diagram showing the amount of cyclic AMP when various concentrations of PTHrP (17-341 NHz) are added to various concentrations of PTHrP (1-341). -34)
and a diagram showing the strength of specific binding ability of conventional antagonists to receptors. FIG. 11 and FIG. 12 are diagrams showing the therapeutic effect of the therapeutic agent of the present invention on hypercalcemia when lung squamous cell carcinoma and pancreatic cancer cells were respectively inoculated into nude mice to induce hypercalcemia. ←-C flashing (koro-Q) 1r Ligation PTHrP (A) Fraction number 2-7 1100℃ 2 minutes ↓ Fraction number 1.8 Annealing ↓ Ligation 1 hour ↓ 10% PAGE ↓ Extraction ↓ Ethanol Figure 8 Poor! Knee! 8 Kaku? 58 Jigoni Kumojo Gobi (JCJ)
(J , −+(JL5(J −1cj(JLI
FJCJ C+) (Ri8H people δ order 5E3≦5go; 1 yo 翳PO YO E54 Tsumu Ripo ligation ↓ Transformation (JM107) Revised ligation ↓ Transformation (JM107) Mouth PT) lrP stimulated Shioto f activity Inhibition 2 Ligation ↓ Transformation (C600) 6'1 Specific binding calcium (Machizodi) Calcium (Machizodl)

Claims (6)

[Claims] (1) A polypeptide that does not have human PTHrP activity but has an activity that antagonizes the physiological action of human PTHrP or a polypeptide that has human PTHrP activity. (2) The polypeptide according to claim 1, wherein the polypeptide comprises the following amino acid sequence. [There is a gene sequence] (3) The polypeptide according to claim 2, which does not contain [there is a gene sequence] which is an N-terminal partial sequence of human PTHrP on the N-terminal side of the above amino acid sequence. (4) The polypeptide according to claim 2, comprising the amino acid sequence according to claim 2. (5) A therapeutic agent for calcium metabolism against human PTHrP, which contains the polypeptide according to any one of claims 1 to 4 as an active ingredient. (6) Transforming E. coli with an expression vector that contains a region encoding the polypeptide according to claim 1 and is capable of expressing the polypeptide in E. coli, culturing the transformed E. coli, and culturing the transformed E. coli. The method for producing a polypeptide according to claim 1, which comprises recovering the polypeptide from a product.