JPS6185399A - Tnf-relating peptide - Google Patents

Abstract

NEW MATERIAL:The compound of formula (R is H or H-Tyr) or its salt. USE:Hapten for the preparation of an antibody useful for the purification and determination of TNF (relating protein). PREPARATION:The objective compound of formula can be prepared e.g. by protecting the amino acid of a C-terminal amino acid, converting the resultant compound of formula Boc-Ala-OH to cesium salt, reacting with a chloromethylated polystyrene resin to effect the bonding with a solid carrier, removing the protecting group, reacting with the next amino acid having protected amino group according to the amino acid sequence of the peptide in the presence of a condensation agent, removing the protecting group from the amino group, repeating the above procedures to obtain a peptidyl resin, removing all protecting groups from the product, and separating the obtained peptide from the carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the production of 1NF
We are interested in novel peptides related to is factor.

Conventional one-ball, two-yama, one-no-no-sou J [Carswell et al.
alu+etteGueri++, 11 CG)
rtdtftshi1. : Mouse, 14111''l, 1
Ndoto, throw ~shin!ノ → then, 211. 'After 1 minute 1,
of f)^mon (J, for 1 cell (,410II,
'4 Kin 1! K
tor, TNF) and naming number (proe)
, Nat.

Acad, sci,, UsA, 72@, 3666M.

1975]. Green (Q reen) 611, soil 111F uii was partially produced by fractional precipitation with ammonium sulfate, gel filtration, etc., and the molecular weight of the above TN+2 was reported to be approximately 150,000 (Pro
e, Nat.

A cad, sci, lJ S A, vol. 73,
381 pages.

1976]. Then Mattius (N=I a)
ws ) and 1 yu, I threw BCG at 1 tsugi, and 2 weeks later I used 4 herrings, and TNF+: #! arise,
Manufactured by tJ and has a molecular weight of 39,000 by the glu i+-filtration method.
Then, polyacrylamide gel electrophoresis (Polya
crystal get electrophore
sis, PAGE) reported that it was five-c67000 (Br, J, Cancer
.

・12 volumes, 416 shells, 1980). Historically, Haranaka et al.
TN in mice and Urigi using ni-bacterium+ acnes) and endo l-4 syn.
F is produced, and its molecular weight can be determined by gel milling method and PAGE.
reported that it was 39,000 (Japan Clinical, 40 Tani, 1872, iQ 82 iF), , L 1
．． :、Low molecules in history - Noshinoshi dark blue color C [Special lid
Showa 5]ri-118714, 1[1]Ei 9-'I
1871 ri>4, same b ri -118-/16
Please refer to the publication].

] - As mentioned above, INF is the core 1 due to the physiological activity of t. Jinj1 also contains animal blood and protein component C of mountain rice.

- Attempts have been made to produce similar physiologically active substances from human cultured cells, such as amines (AM).
I NO) et al. 11, Hi 1 - Cultured strain 4 III cell lvl 7002 'g Ha 13 7001 Ni 7 force r Ngunmamelekbun (1) IIA) should be avoided (this J, Su, Mouse 1 - 44!J (rJ soluble prisoner (It l 'l'-I C
L ) induced production+: bright color (T'l+c
J o n al or l mmunolou.

Vol, 113, N(+, 4.1334-1345
('+974)) According to the report, the above is possible.
Molecule of d sex factor is 68000-150 by gel or overpayment.
The range is 00(). They are Rin・1\1・
-1-syn (Japanese Unexamined Patent Publication No. 50-84827).

Alternatively, proteinaceans with anti-cancer effects (Japanese Patent Publication No. 2003-111012) 1]
No. 141519] It is known that there are 11 treatments for malignant Il!4 tumors.

The invention attempts to solve the problem - ■] Sosei Komotobu and Akira are the l^ of the above-mentioned TNF (1'Nl-related protein).
To provide an antibody that can be produced and measured, to provide a specific TNF-related peptide that can be used as a barbutene for the production of the antibody, and to use these to provide the above-mentioned TNF-related peptide.
The purpose of this research is to provide technology for producing 14 F-related proteins.

Means for determining the point of view According to the invention, the general formula %formula% [In the formula, R represents a hydrogen atom or t-(--ryr-day.

] A specific TNF-related peptide and its salts are captured.

At Water Gate 4111m, abbreviations for amino acids, Bebuti 1:, protecting groups, active groups, etc. 1.1
l tl I' A C1I (J B's prescribed formula I
For the time being, the field JJ (following the Jru 1 - usage symbol, the IA of A is listed below. Yo IJ)! Regarding Mino Pass, etc., there are 11 C optical anomalies, and it must be specified in particular. tjN!l'b u').

Ala: No'Lan I-eu; o Iren 3Q
r; L phosphorus Asp; asbafugin lys; lysine pro;
Tertiary-buto-4-dicarbonyl U[-311; Benzyl group MLSll; 11 Me-1-4-dibenzyl 0[:3
Zl; Benzyloxychloride CI 2-13zl: 2.6-diku1]rubenzyl Cl-1;2 Chlorbenzyl A4-dinonorbonyl The compound of the present invention is an easily available commercially available aminohll'
t4 Easy to synthesize with 11 simple operations/J'
-5= Yes, use this as a hapten - CfA epidemic I/I11
;i can be prepared, and antibodies having specific reactivity to TNF can be easily and stably harvested from the antigen. The specific antibody can be used for the production of 1-N-related proteins by, for example, binding it to an affinity chromatography antibody and using it in the chromatography. It can be used as a specific antibody in immunoassays. In addition, the Bebuti of the present invention can be used as a labeled antigen specific to the T'NF-related protein by adding various materials to it, and by using a secondary specific antibody and mW antigen. , 'T' NF can be immunologically measured.

Hereinafter, the chemistry of the peptide of the present invention will be explained.
Ru.

The peptide of the present invention is a conventional peptide synthesis method, specifically ``The peptide (1'l+a l'eptidcs).
) J No. 11 (1966) (3cllrode
r and L ubkc@, Acadeiuic
Press, New York, L) S
7F) i4-)), for example, the ajiF method, chloride treatment, b! I Retsuui Monota, U acid and 1 & 1 method, l) to C Buddha, active ester is (1 noni 1
0)lnil1.1. Chill method, N-Human El 4-C”
[Methyl] containing chloride, cyanomethyl ester (removal), method using Wood-Node reagent, carposis midazole, redox method, D CO, -7
Ding I Ryoi J (110N 13.11081, l-10
Su)? Prices include 1 depending on the person. In the l horn method, both the solid phase occupation method and the r wave phase synthesis method can be applied. According to the method of making boribeno f I', for example, add 1-))1'% sequentially to the terminal amino scissors and add Ji4 A
From the end of the WI4 class/works, × is divided into several noragments and cut.
ノ(I< /J) leave (gomari is manufactured).

For example, the solid-phase 8-composition method is omitted for details! 1Φ-divination, 0
Terminal)'Mino Fiber ()'Mino M 4rI81! I did it (
J)) -bi-methyl 1ζ1 t, LJ,]
So, yofui111-1111L l go 9 a t!
6゜Fi/7 Il, ut body Toshi (ha, k mustache, 11
h Le Elementary School 4 Shiro Domusubi & 1] Including i! There is no limit to the number of materials used, for example, chloromethyl resin, bu[1-
A tert-methyl resin such as a t-methyl resin, a hydroxymethyl resin, a phenol resin, a tert-al4yloxycarbonyl hydrazide resin, and the like can be used. Then, after removing 4i+ of the amino-protected-M, the amino-U-protected amino fusions were sequentially added according to the )'minoMv series represented by the above general formula (1) to form a condensation reaction with the reactive amino group and the reactive carboxyl group. After combining U, one step - f and synthesizing the entire sequence, Bebuti 1! By separating the compound from the insoluble carrier, the present invention can be obtained.

Each amino-chou that has 1l11 functions in 15 methods of the 1st era, such as 1y1゛, A sp, L yS, If
is, S er17 protects its side chain functional U (if this is the case, it is protected by a protective group, and after the reaction is completed, the protected U is removed. Also, the reaction ( Nikan': 3 Ll's automatic failure is oil-tight and old.These reaction fj methods are well known. J-ba is interpreted as appropriate.

For the protection of amine forests, examples include benzyl-1-cycarfol, 130G, [er[-tunomyl J-t dinonorbonyl, isoboroul Al-dino'rubonyl, 1)
-1-4-dibenzyl A4-carbonyl, CI-Z,
Adamantyl A-1, Cicalbol, Triful lJ mouth)
Examples include small sulfur, notaryl, small lumyl, 0-21-1-phenylsulnol, and small sulfur-notaryl crystal.

hrubo 1nru foot l! l! For example, Kensaku Doji is Al 4.
, ru1rru (methyl, ethyl, propyl, methyl, t
full 1-ru 1 stell such as arI-ij le), [3zl-
r-STR, p-” l-[1 benzyl ester, M
Bzl ester, l) ri [1 [1 benzylnisder,
)＼Zhydril 1, Stell, Carbopenzogishihitoraj 1! , terl-butyl A oxycarbonyl hydrazide, 1-lythyl hydrazide, etc. are exemplified.

The 3er hydroxyl U can be protected, for example by esterification or esterification, but such protection is not necessary. For this esterification, lower alkanoyl groups such as avipur, alkaline esters such as benzoyl, etc.
-frunoM, benzoyl'A4-cyclocarbonyl, 1-tyloxycarbonyl, carbon resistance of 68, etc. are 2I
can be lost. J, ll, 1-nor f1. I m Zuru base return 11, Bzl, Tetonohi 1; ++ Pinol, t e
rt-Butyl M@ is given as an example.

Examples of protective groups for Tyr's water legs include λ-ba1311,
C12-BZI, Benzyl A4-Dino J Rubol, No/
L Chill, TosM, etc. are rejected.

The amino group holding yIM of LVS includes benzyl A xino J rubonyl, CI-Z, CI, -Qzl, 13
oc, T 08M, etc.

Protecting groups for the imino group of 11S are t, ros, Bz
Examples include l.

This is carried out by esterification of ASp with carboxyl alcohol, methanol, ethanol, +ert-butanol, etc.

Examples of activated carboxyl group b include the corresponding acid chloride, fermented anhydride or mixed white anhydride, azide, active ester (bentaf E-phenol, I]-nitrophenol, N Di1゛(]4: silixinimide, N-hydroxybenztrinosol, N
Human 11-1 5) Norborne]/? , 3 Nka no tobo 1]・1 class with r mido etc.) if/r' lgera 11ru.

I / J N person (ni, 1 mei (anti-1.L,
11 Tsunomi] Top 1 and It tb l:, t
Horn 71 E) Ha with Sill U Old g L+:, (
P-I tide combination + January 1st heat (1, 17T's (f
(+l-f, -, B < book) Hru. id medium and C are base/f I: iris formation (, 4 important things to know about
ζ-(8 camphor trees, e.g. λ is on the left (water, l, etc.) )), pyridine, chlo[''form, dinoj1 phosphorus, diltU lumethane, Ttracht1''nofun ('I II+), hl
Acid 1. Methylpyrrolidone, N Methylpyrrolidone, (IINIf) is a koma (, usually, but not limited to -h
(Kome) Shikusen IJ (・Ishi Rumi) 111 sills
1 preferably equal to 7 rue −1,! NIL le woman and 46 years old. The range of reaction used for the reaction is generally about 60'G, preferably about 20'[ ;-about 4
＋）' is selected as appropriate from the range.

Reaction time 1.1 Generally, it is about several minutes to 30 minutes.

Mixed h! Anhydrous V, J is prepared by adding methyl taro[]M acid, mebmethyzo 11, methyl phosphate, methyl chloride, methyl ester, methyl ester, methyl ester, methyl ester, di[11 -I IJilm, (I become a compound using an al-4-halocarboxylic acid such as 1f1-li]-thyl), I
・Limethylamine, pyridine, dimethylj'niline, N
-Methyl phosphorus, 1,5-cyazabicic r+ (4
,3,0) nonene-5(1)BN), 1.5-diarydischif(ri,4゜0〕untesene-5N)I3LJ
), 1, 4 Jino'riji shift tl (2,2
, 2) 4 Kuttan (1) to 1300) etc.
Rekkoku salt U such as X'1 oil potassium l1,1fuchu Jl-lium, carbonated aqueous potassium, carbonated aqueous potassium, etc. can be used. Solvent J-shi (is ltI!t! 1 Each yellow solvent for 1-membered method, specific 1: is 1-chemical method / 1. Non, ri [
I [1: Iil, Lum, Dichloro1. J-1 hydrogenated hydrocarbons such as tan - A class, benzene, 1 to 1.
T-thyl compounds such as 1-thi[non, etc., diethylnithyl, 1111-, and lame 1-4nidiethane,
1. Solubility, DMF, 11.・+80, II M l-) Use a non-7111~n polar melting wax such as A.
The reaction IJ'1bd is generally carried out for 5 min.~ + OIIA lid, preferably 5 min.
iI'('There is.

In the cytation method, first activated nonorphoxyl U, alcohol Cic! such as methyl alcohol, ethyl alcohol, benzyl alcohol, etc. i 14, l, dicarbo71sil, hydrazinho4'11
The reaction is carried out by reacting a substance in an appropriate medium (e.g. di4niline, l).
h+ F, l) NII s □ or any mixture)
[Use a solvent etc.] (,5).

The use of ditonodine hydrate is activated)
Sildan (, the amount per unit is 5 to 20 8 moles 1 preferably (ma;) to -101 8 moles h\"2 5 to 1
6 (1 submission! + ('l' must be 1, preferably 2)
0-・30′G-((i rope 4 mark. 1tli <
L/ ”(Nonorbo 4.Sil MIJ force h(J1;Fujin(u1φSare Nogo 1118 IJ (Jidrash 41 bodies)
Give 14.

The carbo-4-syl group N1 is decoupled with azide.11/This compound is prepared in a suitable solvent in the presence of an acid. By reacting the hydrazine derivative obtained in Section L with a nitrous acid compound.
:Remanufactured. As an acid, I usually use j-ken. As the I medium, dioquinone, DMF, DMSO or a mixed solvent thereof is used, and a nitrous acid compound is used, and -C is nitrous acid.]
・Rium, f-soamyl nitrite, di-rosyl chloride, etc. can be used. Such a nitrite compound is usually used in an amount of equimolar to 2 times the hydrazine derivative, preferably equimolar to 1.518 molar, and the reaction is usually carried out at -20 to 0°C, preferably -20 to - The reaction is completed at -10'C, generally within 5 to 10 minutes.

Incidentally, the peptide bond forming reaction can also be carried out in the presence of a condensing agent such as dicyclohexyl carposi, rmid (1) CG), carbodiimidazole laethyl pyro small sphine, etc.

In each of the above reaction steps and M final step, h1 of the protecting group
When 2-elimination is hardened, this follows a normal elimination reaction -) (
11', i) Jr. As a reward, open lambda buff.

"Using 1thl, such as 11 black,
Itoyika, Liquid Norn (Ni 7 Medium Metal J Thorium Earth Reduction Guard υ) Un几的f) Buddha, Llinor J++alal, IM
(U J: Nt =, Kuwa Mizuki, Men9nsuru Konno, Attenuation) "I would like to show you 1, 2, 1, 2, 1 Kutsu' Citrisis, etc.

1^1: Addition of water-A using a touch M is, for example, a ho yarn 111
Me (IF, 0. to 40'G (ti not (4). For catalyst 11
The reaction ends. In addition, the above-mentioned horn cytolysis is usually 0 to 30' in the absence of solvent, preferably 0 to 20' (:
Approximately 15 power at about lj1 I It! 14 schools 1Ij431
i! l, (b).The layer used for acid is To(>A
11 @ "Usually 5...10 times more than +ii! Is it about 11 more?", no. In this acidolysis lJ, only the 5A-U of the amino group is removed! J), it is preferable to use trifle A-[J h1 fit or IM Mizuki M with scissors. Ten il+ for history! Liquid An ni Nino' Medium Metal) 1~
The speed source using Rium is that the Ryo liquid is applied to the bar 1 nonent soru for about 30 seconds ~ 10 minutes 11 minutes, and using the metal no 1 - Rium of Unakushi, it is 40'C ~ -. 70℃
At Choseki (I don't get it.

The bepide of the general formula (1) produced as described above is separated from the reaction mixture by means of separation such as extraction, distribution, columnar column [j7 togranol-'d, etc.]. Ru.

Thus, T N F of Honkomei expressed by the general formula (1)
Obtain the related Bebuti 1ζ.

This is how you get 1 bell! (1, 1251 for this,”
'l W17) f+RIJl'lvA trade, bar A
4 Jitter (! (POX), 4:Mol ~ Ribsinogen, I l"1 Carboxybeb J Dase, Gly[11)'
Runo I: 1! ζ) Phosphorus Mhi2 Mizuki enzyme, amine-1! , Kosuhorino 1! ,0--Nase,P
-Name, β Sword Noctocida-+i, Glucose-
6 - Fuji Ai lEnononotsuLi(R
IA)) person or
) used as the IJ11 recognition antigen used in the method (・
Monkey. Introduction of the above-mentioned 11i
: Really fruitful! ! can. For example, the family Ω1 property "-do is ri1
Oxidative iodine conversion method using 1nomine 1
, If unler and F , C, G
ree++wooJ; Naturt.

-1ri--4, /+95 (1962), Bi
ocl+eII1. ．． 1゜L Gai, '1471, (1
963)], etc., and the introduction of the intoxication test tube is an example of the usual horn-tubbling method, λ1.
F,hi1ial(jar) la,J)h? Man 〔
△ eta, E ++1iocrtno1. Su
ppl. 168 1206 (1ri72)) and Rikimeru (M, lI).

1ri ill'01) /] >h (Pro
e, N atl, A Cdd, S Ci,.

(H3A,,!t-/,-,713(1067)) Hiron's public knowledge 0161 people 1, ゛yo-) ((r', c) can be done.

1. Manufacture of hooks using Honmaru Akiba 1F as a hub ten.

The antigen No. 1 is represented by the general formula (1) c-↓′Ja Ming・,
Let /f1; be Ha/Non, and this is Habte] -11 The θ in the test sample is reacted with an appropriate carrier, and J and S1 are converted to 1. In the 1st century, 111 bodies were assembled in Jj I-C Hand No. 7 (7), which was usually used to create antigens in 1&4. Kaiden is used (3). Among these 10 bodies, for example, a horse +
a + r-eye Alnomine, 21 blood 11] '7 J Min, Urigi 1bf + z' Runokushi, Ji 1 Blood 1^)) Luf Min, Jitsuji Serum Nor'l/Minze, 1llI! l &l's blood 2^ no'ru 1 min knowledge: horse blood) ^g [1 norin,! 1 blood ng^g 111 rin, 1 shiri t^g [1 norin, hi 1
□1j11＾g117rin, jitsubu+/++ i＾gl
''Purine W's 1υl/+ Serum Order I Phosphorus ;), Anciformes Order I Phosphorus,! 4j1''n II 7 Rin, Uri 1 de Bu11gl1l-/Rin, Hi l-j ++ri1j Purin, Jitsujichi11g II'/Lin etc. -) 11 no 11 '71Jn; , Haru no ＼ [ri 1m/rin, cow 7g 11 rin, urigi heshigu [-IJ pudding human hemog] 1 lin, sheep baleen I'l 7 rin and other animal fins ■ Norishi; 4 --Small animal hepatinone'- such as Lurinbet I\Moshinone
Ascaris J, extracted from IJ protein trade (Ascaris extract, Japanese Unexamined Patent Application Publication No. 1999-11641, April Publication, J, 1111-old), 1 'I 1.2
(3(1・~268 (1973), J, 1mm
Old 1. ,122°302~308(i 'J -/'
, ) ) , J, l v+on, 娨-needle.

893・-900 ('l 9 (3-/ ) and Am,
J.

p l+ysio1. , l-Ewa-4)'/h-E)
78 (1') 60) J1/,: b's or ni, etc. are in the history for a while! Koshino).

Small lysine, polyglutamic acid, lysine/noglutamine co-m1 complex, lysine or ornib]/co-containing complex, etc. can be obtained.

Among the body-containing reagents for Habten, there are a wide range of substances that are considered to be one member of the 1) function of passing antigens.
(゛kiru.

Specifically, 17+', l-1is is cross-linked,
For example, bisdi7zotized benzidine (BDB), di7zotized benzidine ([3[)
I)) Diazonium compounds of '4; avoid cross-linking between amino U and amino U, such as glyokyl, n[]n dialdehyde, geltar aldehyde, aliphatic dialdehydes; cross-linking between thiol U and thiA-U, etc. to bond, for example, N, N'-0-1 nylene dimerimide, N, N'-lit-phenylene dimerimide, etc.; amino, etc. are crosslinked. , for example metamale r midobenzoyl N-hydroxysuccinimide ester (N.jBS
), 4-(Male-midomethyl)-Schiff1]hexane-1-carboxyl-hydroxysuccinimide, etc.
-Ciscusine-r-mide T stellaniamino group and carbo-
M mulberry, IA, etc., which is used in the usual peptide bond forming reaction to form an amide bond with 1 sil U, and IA, such as DCC. N-, T-thyl-N'-dimethylaminocarbodiimide, 1-1-thyl-3-diisopropylaminocarpodiimide, 'll-
Schifhexyl-3-(2-morpholinyl-4-■thyl)
Examples include i2 hydration agents such as carbodiimides such as carbodiimide. Also, 1 Hab Ten - 10 I ↓
As viscous reagents, 1) diazonium such as -diazonium phenyl 1 enzyme, Noryl JJ Rubon b1 class and ordinary peptide bond forming reaction reagents, such as the above-mentioned dehydration bond Thl.
The union t! /． : b no L] Shitagawa 1iJ is capable.

The reaction for producing the above-mentioned immunogenic antigen is carried out in a conventional buffer solution of λ, water), 8 oil or i+ 11 7/-10, preferably 11 1-+ 8-9, 0-40C. , good,
It will be held nearby. The reaction is typically 1-
・Complete in 1ml of 24suke, preferably 2 to 5 hours. I
-Fit! Smell (typically used as oil resistant):

0.2N Water M 11-Notriu A-0.2Nl Small Wu 0
, 2 N・1 珈 1 hino j ri ・”] 113 No. 1
Ika-■n 1k, 0, 2N・1 Norium carbonate-0.2
M small □. 2M Current IL Power Liu 8-1 Oil, 0, 0 巳) N・1 4-Hochu J Thorium-0.2N + Horium 0, O ji M Notorium Chloride Loose 1i Oil, 0
, 1.1 phosphoric acid-71(-A)j-shim-0.05N
Four M notorium slow-smelling [smell (ha/ln, ha1 ten-damage ligation reagent and one
The use of 0 bodies can be determined as appropriate, but if λ. 1 t, (111 bodies 1 = 6 f
8 coniferous culm pox, preferable (〕<1.L l −= :)Iro−1 degree, and habten-losing body binding reagent 1−′+0
11! ! It is best to use about 1-E.

In the above reaction, the desired immune antigen consisting of a peptide-111 complex bound to Jf1 body and hafrane is produced without using the J, River IT-injured body ligation reagent.

The antigen obtained after the reaction is processed according to conventional methods, such as dialysis,
lIIliI can be easily obtained by gel filtration, fractional precipitation, etc.
II pongee can be made.

Using the immune anti-11λ obtained as in Kazuki, the method for producing antibodies is to inject the above-mentioned antigen into mammals IIJ,
The desired antibody is produced in the body, and this is removed! lru(
: Implemented by.

The mammals J: L and C used for antibody production are not particularly limited to i and II, but it is usually preferable to use 1- or 1-. For antibody production, dilute the prescribed amount of the antigen obtained in step 1 with physiological saline and add Freund's auxiliary solution (CompleteFre).
und l s adjuvant) and combination t,C
If we prepare t+mn and administer it to mammals. For example, the Milil solution described above is injected intradermally into a rabbit (0.1 to 5 mo/time as the antigen eel), and then 2 31N 1
The immunization may be carried out by administering each gl for 2 to 10 months, preferably for 4 to 6 months. Antibodies are collected by removing blood from the immunized animal 1 to 2 weeks after the final administration of the -[&] suspension, centrifuging the blood, and dividing the blood for 4 minutes. According to the above, antibodies with excellent specificity for the used immunization antigen can be obtained, and can be used in RI and IA individuals, etc. I liJ L, ('T I
``Identification of J F bLI-linked protein-1,- (electronic Iru).

Practical example 1 strike, book 1 light 1 length, 2 h 1 shi<explanation lJ,
4. 1 of 1 bJ, an example of the preparation of a precursor antibody from the 1-nod of the above-mentioned antibody, and 1 of the antibodies from the above-mentioned antibody 1 of 1.
However, this light is not limited to these. In addition, in the Ryo-komi example (Juru R t &t+ is 5・su/J Guru 10 I'il-小目一、ノド'l / / (-L: (1
-L:III! A! Ml! Itkawai (L for igllIM)
, /= Shi no guru,.

Rf' -+1 Tutanol Pyridine 11￥1
school ・J, (i: 1: 'l: l) 1<12...11 ivy, nor pyridine [1i V
) j, < 30 : 20 : (i : 21
)〈Bell-do〉 Manufacture lI41 1 (1) 1. I Lli; A Ia O11!18
71m1J to 1tanol 1()-+U or I, 'Shin,
Add 7 AWI water 3-. Therefore, 1) 11 meters 4 used () Gosium High i: I-1 Ken Carboneton? 1) It becomes 11-1.0 (1Al). Transfer this m solution and dry it [30C-A 1a-1:
Obtain sium salt. This and 5 g of 1-meblated poly J-ren resin (+44 Protein Trade Research Promotion Association, 2% divinylben U, mesh 200-400) were mixed in DMF40.
In addition to -, using a magnetic stirrer, -k, 50
'C stir the 4 resins I) N=I +, January vl
F, 'Ha O(9: 1, v/v), l
') Ml- and ]-tanol in this order, thoroughly washed and dried [1oc-A la -6111i
i 5, 2 'I li 4 +! Fru.

After partially hydrolyzing amino Mj″IU1 71 <i”
/, -Bundle)'Minoba0.25mmmoji/(l 01
1m ('A:) /, -6tgl J: Boc-A 1a (allllt 2, (')(
+ Nori[' D Form 30 J-C After washing 3 times, Ri09m1 Linol A [j1 Ken (-1-F A ) Nori CI I-
] Add 301 J of Dolmun l solution, until ) - and 2
0 minute reaction 1, tree 1) 1ori 1111 horno,
30- once, methylene chloride a Om-<1) times, 10
9. Methylene chloride of l-ethylamine 130
-["3 times, then -C' j-methylene 3 (1wm
(Rinse each time six times to obtain H-A la-resin h.

271 u of Boc-Val-Ol-1 was converted to chloride Me'/
Add 20- of liquid U (dissolved in l/n) to the H-Ala-resin described in L, then add II -React for 2 hours. After washing the resin six times with methyl chloride, I'3o
271 mu of cVal-011 and 1-human rl 4
-Cybenzotrinosol I 91 In methylene chloride 2b-1 1ullλ1, then ('l') 2' of C□, -> 8 8k 5- IJII E C again (reacting to Su (-katsuno ° ring payment).tit 1lli
JL conversion me/-1, non (clH-ai'l, (L
locval-A Turn on the 1a-4Bl side.

(al.(2)), Boc-Val-
11 of Ala-W fat
]' Mino Chumata (Yoriruho 4-sil L (σl 1lls)
l111-dlJ]'mino11i2 is sequentially condensed and m
113oc reaction (J).

Mouth OCVi eyes (')If
2 7 'l + usu1 (oc 1lis
('Ios)-Oil 5 1 l
llIsu11oc Ala Oll
236 Lliu130 l Qll (Nl ・++2 0
3 1 1 W+J13oc-1] ro
Oll 269
-〇Boc-Lys(CI-Z)-011519m□[
3oc-Asp(OBzl)-011404m.

F3oc-8er (Bzl) -0ff 37
0wυBoc -Leu -011・l-+20
311 mLJ [3oc-Ala-Off
(236mυM4j coupling reaction)
11-8 - Remove [3oclJ, dry, I
I-Ala le eu 5er (Bzl)-ASI
+(OBZI)-kawa-ys(CI-/)Pro-1e
u-A la-II is (choos) \/al-
Obtain 2.99 of ValAla-Ml fat. Gono back 50
0111 (+72') -le 1.5! and fluoride water lA
10m1. , : Ia, -20'G - (-30 minutes, then O'CT' for 30 minutes, after avoiding the ink 1 bath, excess fluoride water A (r) was distilled off under reduced pressure, and the remaining 10% The aqueous layer was washed with acid (J-clIlIl and 1'-il), and the aqueous layer was lyophilized. Kagashi, 14-speed model 11-to-gura 7-1' (t
lPLc) (TSK'/'le 0f) Sl 201-1
4.6x25.0+n, Nakabei Kaidat Co., Ltd. J (Company '14:
in lit h, AaC = 0.01%I l-A
2&H 13i1 to 261 = C113 to N to J, gradient (0 minutes to 10
0%, 5 minutes Part A'100%, 15 minutes - Tip A 85)%,
4;) minutes - 45% and 50 minutes - 0%)
la 1-OLI-ser-ASu-t-VS-P
ro -1-c++-A la If is -Va
Obtain 35+au of t-Val-A 1a--011). Hereinafter, this Bebuti 1: will be referred to as "Bear/-1'AI."

r<r: Rr '-0.69 Rr 2-0.30 Amino acid fraction digit value: (divided by t-1X'L835 type)
Minute digit value As++ (1) 1, 09Sep (1)
0.95 l-)ro(1) 1.04Ala(3)
3.00 VaN2) '1. Q6 1eu(2) 2.10 1 Vs('l) 1. 'l 411
is(1) 1, 03 The upper amino acid analysis values are the results measured after hydrolysis with 6N-hydrochloric acid, and C is also the same in each of the following.

ll! lJ Production Example 2 tl-Ala-Lcu-8er (Bzl
)-ASII(OBZI)-Lys(CI-Z)
-Pro-Leu-Ala-1-1is(Tos)
-Val-VaiAla-500 WIJI of resin,
Boc-T to 1tiJ Ji using DCC43mo and 1-human[]4nidibenzo-1-heliazole 32111.
The target peptide (II-Tyr-A la
-1eu -5er-Asp-1-ys-1)ro-1
eu-A la -1-1is -Val -Val
-A la-0f-1) 30111O was obtained. This peptide will be referred to as [Peptide DJ] below.

Rr (m: Rr'=0.75 [2=0.28 Amino acid content 41iIIJIJ:Nj tachi83;) analysis) Minute UNm Asp(1) 1.07Sar(1)
0.991)ro('l)
1. (')5Ala(3)
:3,00Val(2)
1.981 (II1 (2) 2゜ 011'yr(1) 0.951
,yS(i) 0. 07l-1
10a of the peptide A of the present invention obtained in Production Example 1 of is('l) 1.01 (Preparation of immunizing antigen) 2I> Be II1:
+ u bovine blood)^1 Rubumin (BSA) 20io etc. is dissolved in a small amount M--i+4f (0,1M, pt-18,0)2-. Add 0.1 mol of geltalaldehyde ifJ solution to this solution and stir for 5 hours with no leakage.

After that, reaction i11! The mixture was precipitated into 1Q of water at 4°C for 48 hours. During dialysis, the water was changed 5 times, and finally 11
! l! Dialysis against 1Q of good salt water (-). Next, the total amount of the 44-d solution containing the peptide-protein complex 14 is quenched with physiological saline to obtain an immunizing antigen solution.

This antigen has an average of 20 moles of peptide A bound to 1 mole of B5A (with a molecular weight of 61,000).

Preparation of Antibody> The M suspension prepared by adding 3- physiological saline and 5-
nd v old te rabbits, 2. 5～
Administer subcutaneously at 3', 0 kO) and then every 2 weeks at 5
The same amount as the first dose is administered once, and then every 3 weeks thereafter for 3 times. Seven days after the final administration, whole blood is collected from the test animal and centrifuged to obtain antiserum (antibody).

Preparation of 0-labeled peptide Peptide B obtained in Peptide Production Example 2 [ ] Lamin T
The method using + [changes as follows.

That is, 0.5 molar mW of the above peptide 5μ0 (
pH7,0)2(lQ with Na (+251)(Ca
rr! 0rrl'ot! N, E, N, ) l
,: 0.5) molar phosphate edge of 11-4-4-II-
欣20tt *To tell 1λ, 〕giIIri 11〕/1
70Illt1 1's O,'>Lノ1.11-N deception 2 0tt Q tlAl get. To-(・
30 seconds 1-anti-k 1 (meta IJi! 11! --)
1-lium (Na i S, 0.) (i(lIIl Ri)
- No. 05 Kami Lulin Oil Yuru 1 Pass] OI and Q (reaction is 11 i3 t!).
-] Add 100zzQ of I.lium aqueous solution, and pour the reaction mixture into Lenor J'Tux G-25 color! , (1,OX
:)+01. Elution h: 0.2596BSA, 0.9
%NaCQ and (0.r containing 10,02%NaN3)
5 Shiru Rini/Long resistance klura, i+ If 7, 4
) cyiVL, '<+251 (゛Video lJ bezotide B)d.

Measurement at zero force 1111 The titer of the IL antibody is measured as follows.

i.e. anti-llI 0.2% t3SA, 0.005
95 Tweet: / 20 and (Fo, 02596Na N3
Including 0.017 Lurinchu Mmf+& (++ If
7, 4 > 2(), 60.80, ri 11 o, I
620.4860.14580.43740...
...double the excitement, and each of these 200
To μQ, 125 l-labeled peptide (41
i-identified peptide diluted to about 10,000 cp-) 20011 and 0.01 molar
Add Wt 200 tt Q, ink 1 base of 24 strands at 4"0, and combine the generated antibody with 1251 camphor peptide by 4 strand-rIf+1!1-deposition method and centrifugation 51-1 method. (4°C13() minutes, 3000r
1251 Minutes Mt, from the camphor-peptide (pHl), the anti-14 (one door 251 The binding rate (%) with the labeled peptide is measured.The vertical axis shows the binding rate (%) of the antibody with the 1251-labeled peptide, and the horizontal axis shows the binding rate of the antibody (%).
Take the rate (, initial 1! le mth) i, and calculate the rate of each mm odor 4 /11 1~! Ill.

The dilution ratio of the antibody at which the binding rate is 50%, that is, the antibody titer is determined. As a result, the antibody titer was 1,750.

Cross test with mouse TNF and human)-TNF-related protein and other antibodies As test samples, the following TNF was used, and I#l semi-diluent L-co, 2% BSA, 0 .005%”/(-
20 and Ol(') 25% Na 0.01
Use mild phosphoric acid (pH 7.4).

Urigi T: Method of Run et al. [, J.

l 111+uunolouy, Vol, 1 25
, 1 671 -167-/ (l O8(')
)), 1960
well ) et al.'s ij (1' roc, N
atl, Acad, sci, IJS
A.

Vol, 72. No. 9.3666-3670 (197
5) Prepared by ), '1.04X10bLl'
d Human-I N + -related protein; JP-A-59-'141
5101 public Qu i! lkl! (7) h? A
II,), t) M-made shield, 5.13×1 ()'
jl ,,-' d In each test tube, various concentrations of IIl, trial 44(-),
2u, antibody 0.2- and 1251-labeled peptide (m-labeled peptide obtained above was diluted to about 10,000cp+11/co(1)) 0.2-, and heated to 4℃F2411Hj.
After dl'n-1-ko-1-shi, normal sheep side)
^(11Bllal=33=sbeep 5eru11+) Add 0.1 mG and then
&0.5- was added and centrifuged at 4°C for 15 minutes to separate the antibody-125 l-labeled peptide conjugate and unreacted (unbound) +25 l-labeled peptide, and the radioactivity was counted. . Total hII capacity used in the reaction (
Self-ratio (B/T) of the radioactivity (B) of the conjugate to T)
The binding rate is determined at each concentration of the test sample.

The obtained results are shown in Figure 1. In the figure, the vertical axis shows the binding rate, and the horizontal axis shows the sample concentration (watermelon of peptide A and each TNF). Curve (1) Hamaus TNF,
Curve (2) Hatsu9+! TNF, curve (3) is
Curve (4) shows the TNF-related protein and the bebudid of the present invention, respectively.

From Figure 1, the antibodies are Urigi-TNF, Mouse-rNF.
It can be seen that there is a cross-reaction +Il with the protein related to Hi-TNF.

[Brief explanation of the drawing]

The first session shows the affinity between the peptide of the present invention and Meishin-1-N[- and the antibody, 1.1! There is a diagram I. (hereafter F)

Claims (1)

[Claims] (1) General formula R-Ala-Leu-Ser-Asp-Lys-Pro
-Leu-Ala-His-Val-Val-Ala-
OH [In the formula, R represents a hydrogen atom or a H-Tyr- group. ]
A TNF-related peptide represented by and a salt thereof.