JPS63501572A - Isoforms of soluble immune response suppressants - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Isoforms of soluble immune response suppressants Summary of the invention The present invention relates to a novel in-type soluble immune response suppressor. high speed liquid By using chromatography and isoelectric focusing, four different biochemical The active isoform (isoform θ) was isolated.

The isoforms of the invention are useful in preventing organ rejection and inhibiting cell-mediated responses.

Background of the invention Soluble immune response suppressant (S-R8) is concanavalin A or interferon Antigen-nonspecific suppressive factor produced by L7-2” T cells activated by children (T, M, Aune and C!, W, Pierce.

1984, 'Mechanism of S engineering RS action at thecelluLar and biochemical 1evel “, Edited by E, Pick, L’Yl! ]pk", Ok ln e S, Volume 9, Academic Press, New York, p. 257). 5IR8 is each Inhibits immune response and prevents cell division in normal and malignant cells (T, M.

Aun e and C, W, Pierce, 1981, 'Mechani sm of action of macrophage-derivedsu ppresor factor produced by 5olable i mmuneresponse 5uppresorta macro phages (produced by macrophages treated with soluble immune response suppressants) Mechanism of action of macrophage-derived inhibitory factor)'', J.

■ mmuno 1. Volume 127, page 368; T, M, Aune and Cj, Pierce, W., 1981, 'Identification and initial characterization of a non-sp ecific suppresor factor produced by 5 olable immune res-p□pS6 5uppressor-t Reated macrophages (treated with soluble immune response suppressants) "Identification and initial characterization of non-specific inhibitory factors produced by macrophages", J.

Engineering mmuno 1. Volume 127, page 1828; M, T, Aune and 0, W, Pierce 1981, 'Conversion of solu bleimmune response 5uppresor to macr ophage derived suppresor factor by pe roxide (a soluble immune response suppressant substance derived from macrophages due to J oxide) Conversion to repressor)”, Proc, Natl, Acad, Sci, US A, vol. 78, p. 5099). According to the recent work of Aune and Pierce ( T, M, Aune and C, W, Pierce, 1981,’ Con version of 5olable immune response ppressor to macrophage-derived 5uppr essofactor by peroxide Conversion of response inhibitors to macrophage-derived inhibitors)''.

Proc, Natl, Acad, Sci, USA, Volume 78, Page 5099 ), E3 engineering R8 is activated with H2O2 to reveal its biological effects (S engineering R8ox ) I realized that I had to do it. Furthermore, the action of S-R8 is similar to that of SH reagent, carbon Talase, p-aminobenzoic acid, ascorbic acid, taurine, or naturally occurring potassium S-R8ox is inhibited by cells by causing oxidation of SH groups in cellular proteins. It seems to mediate inhibition of cell division (T, M, Aune, 1984, 'Mo dification of cellular protein 5ulfhy dryl’ groups by activated soluble imm une response 5uppressor (activated soluble immune response suppressor) modification of cellular protein SR groups with regulatory substances)''.

J. Eng. vol. 1133, p. 899). S Engineering R8Ox is also used in immunofluorescence microscopy. The normal arrangement of intracellular microtubules is disrupted by observation using a mirror, and microtubule proteins are purified in a cell-free system. (R, D, Irons, 1984, 'SOluble immune response 5uppreBsor (S engineering R8) in hib1tsmicrotube functj, on in vivo and microtubule assembly in vitro (possible Soluble immune response suppressor (S-R8) inhibits in vivo microtubule action and in Inhibits microtubule assembly in vitro)'', J, mmunC) Volume 1.133, 2032 pages). Intracystic integration of microtubules and GTP-dependent assembly of microtubule proteins It is known to be sensitive to SH oxidizing agents.

Biochemical structural analysis of the 5IRS protein was also conducted (T, M, Aune , Webb, 1983, +V Purification and par tial characterization of the lymphok ineθolable immune response 5uppresso r　(Purification and partial characterization study of lymphokine soluble immune response suppressant) “1 ．． T, ■ mmunol, volume 131, page 2848; T, M.

Aune et al., 1983, VPurification of racLio- 1abeled 5olable immune response 5upp ressor (purification of radiolabeled soluble immune response suppressant)”, J, J.

edited by Oppenheim and S. Cohen, Eng. .

Lymphokyles and Oytolcines, Academi c Press, New York, p. 383). Early research on reversed-phase high-speed liquids It was found that two biologically active peaks could be recovered by chromatography.

One peak was eluted with 20% propatool (hereinafter referred to as S-R8-α), and the second The second peak is eluted with 60% propatool (hereafter referred to as 5XRP;-β) (T, M, Aune, 1983, 'Purification an d partial characterization of the lym phokine day 01 u’ble immunity response supp resor (’) Purification and partial sex of immunophokine soluble immune response suppressor ”, J. Eng. vol. 1.161, p. 2848).Subsequently, 5IR8- α was thoroughly determined by reverse-phase liquid chromatography using C-18 and diphenyl-modified silica supports. Purified (T, M, Aune, 1983, 'Purificat ion and partial characterization of he lymphokine 5olable immune responses esuppreθsor”, T, engineering mmun01.131 volume, 2848 pages ).

As a result of diphenyl column chromatography, two biologically active peaks were observed. It was. 5DS-polyacrylamide del electrophoresis (5DS-PAGE) Then, the first peak contains one detectable protein of M, W, 14.000. , the second peak contained one protein, M, W, '11,000.

This report did not investigate the properties of S1R8-β.

Experiments were also conducted using 5IR8 prepared using a cell-free mRNA translation system (Engineering, Nowiejski-Wieder S, 1984,' Ce1lfr ee translation of the lymphokine sol ubleimmune response 5uppressor (SlR8 ) anclcharacterization of its mRNA ( Cell-free translation of lymphokine soluble immune response suppressor (5IR8) and its m (Research on the properties of RNA)', J, Eng. 393D 2.6 Shortly release Po1yA” RNA from hybridoma cells and add methylmercury It was fractionated by Garose del electrophoresis and translated using a rabbit red blood cell extract translation system.

RNA encoding 5IR8 activity was recovered in two fractions: one with large 25S corresponding to the size of M, W, 14,000 and 8,000 SlR8. , the second is M, W, 8.000 S engineering R5i corresponding to 21 and 22s 'i generated. This result provides genetic evidence for the existence of two or more types of S-R8. For the first time, we established the possibility of

Detailed description of the invention The present invention has a molecular weight of approximately 11,000 by molecular sieve chromatography. and when assayed with in vitro antibody-producing cell responses of sheep red blood cells (5RBO). 1, OX 10"-7, OX 1011 units of inhibition/μy protein specific activity A novel type of soluble immune response suppressor (5IR8) with IR8-(!6.5IR8-G7 and S-engine RB-β7).

See Example 1 for details of the bioassay used to detect 5IR8 activity. thing.

A novel version of S-R8 protein due to non-invention can be applied to one or more high performance liquid chromatographs. using the Laffey (apLc) step, followed by tonic isoelectric focusing. can be obtained. In a preferred embodiment of the present invention, a novel isoform of the 19IR8 protein is obtained as summarized in FIG.

Each of the novel in-type 5IR8 proteins of the present invention obtained by the above-mentioned method is a granular delta. Detected as a single 5IR8 active peak in the cracking FF (see Example 2 for details). Teru). The specific activity of the in-type 5IR8 protein is determined by the in vitro antibody against 5RBC. 1.5 g of protein per μg of protein as measured by cell response. OX 1011-7. Q　X　 1 [ ] 11 SU (7) Range was torn (see Table 2 for details). 5IR As a result of analysis of the in-types of 8 proteins, all types were M, W, approximately 110DD. It was suggested that the thickness is equal to y.

Furthermore, SlR3-G7, which is a preferred isoform of S-R8 protein according to the present invention, is It has the partial amino acid composition shown in the following table.

Asp: 8.45 Thr: A, 66 Ser: 5.54 Glx: 13.00 G17: 11.97 Ala: 11.93 Van: 8.02 Mθt: 1.87 11eu: 5.69 Lθu: 9.20 Tyr: 2.77 Phe: 4.28 His: 2.5 3 The novel isoforms of 5IR8 protein according to the present invention can be used for organ rejection, such as kidney, skin, heart, It is used to prevent pancreatic, bone marrow, small intestine, and lung transplants.

Individual isoforms of the 5IR8 protein can be used as such, or Mixtures of two or more can also be used. The mixture is placed in a short distance or, although there are several isoforms of S-RI3 protein, There are no non-5IR8 proteins, so the composition is a mixture of 5IR8 protein isoforms. It can be obtained by stopping the purification at a point where the

The novel in-type S engineered R8 protein according to the present invention can be used in combination with conventional pharmaceutical parenteral carrier materials. Pharmaceutical preparations suitable for the prevention of organ rejection can be provided.

The new isoform of 5IR8 protein can be administered parenterally such as intravenously, subcutaneously, intramuscularly, or orally. administered. Compound dosage depends on many factors, including the compound used and the formulation. are doing. Dosing should be done in parallel with current treatments used to prevent organ rejection. I can.

In general, antibodies against the novel isoform S engineering R8 protein, especially monoclonal Alternatively, antibodies can be prepared by methods known per se. These antibodies are known as It can be used for diagnostic or therapeutic purposes and for purification purposes.

The invention is based on the following examples and may be better understood when considered in conjunction with the following drawings and tables: be deeply understood.

Figure 1 shows the purification flow diagram of SIR8 protein and separation of isoforms. .

FIG. 2 shows the separation of SlR8-α and SlR9-β by reverse phase HPLO. Se The biologically active fraction from the Fadex G-50 column was transferred to the Licrosolv RP-18 column. Put it on the mu.

The flow rate used was 2 ml/min, fractions were collected every 4 minutes, and 1% of each fraction was collected. The biological activity was measured.

Fig. 3 N-side of S engineering R-α by reversed-phase HPLC using Beam Chlosolv RP-18 shows. The SlR3-α eluted with 20% propatool on the first column was collected in a reverse phase column. Apply to Rosolp RP-18 column. Flow rate Q, 5+++//m1n images every 4 minutes The fractions were collected and each fraction was assayed for 1% IsR8 activity.

Figure 4 is based on a Bakerbond zincenyl reverse phase column. Chromatograms of SlR8-cap and 5IR8-αn are shown. 5IRS-α I and S engineering Rs-all were applied separately to a Paykerpond diphenyl column; Flow rate 0, 5 ml/min; Perform fractionation every 2 minutes and use 1% of each fraction. The S-R8 activity was measured. A, SlR8-αI t B’S engineering R6-α■ . In A, protein 1n-70 lovanol was eluted with a linear gradient of 0 to 60%; in B, it was eluted with a linear gradient of 0 to 60%; It was eluted with a gradient of n-propertool for each step.

Figure 5 shows 5IR8-β Ricrosolv RP-18 (A) and Paykarpondzi. Chromatography with a phenyl (B) reverse phase column is shown. RICLOSOLV RP -18, the SlR8-β eluted with 30% propatool was reconstituted with Lichrosolv R. Chromatography was performed using P-18 at a flow rate of Q, 5 ml/min. . Collect the bioactive fractions and chromatography on a payer pond diphenyl column. I put it on. In each case, 1% of each surface was analyzed for biological activity.

FIG. 6 shows the decomposition of SlR6-α and SlR8-β by EF. SlR8 The collected bioactive fractions of -αI, S-R8-α■ and SlR8-β were granulated separately. The gel was eluted with deionized water. The pH of each fraction was measured. After determination, 1 μl samples were assayed for S-RS activity.

Figure 7 shows molecular sieve chromatography of the S-RS isoform. Reverse phase HPL C! And IEF-purified SlR8 was subjected to high performance molecular sieve chromatography. Geru. Measure 5IR8 activity for each fraction. Arrow indicates elution of protein standard marker Indicates time: Mouse Egg (16o, ooo); BSA C66,000); Egg albumin (45,0DD); trypsinogen (24,000); and Lysozyme (1,!i,000).

Figure 8 shows 5DS-PAGE of 5IR8-β7 (II) radiolabeled at 125 days. shows. A of 125 neeS engineering R8-β7 after 5DS-PAGE under reducing conditions. Autoradio Durham. The most prominent bands have m, w, ~a, o o o vinegar. m, w.

~30. (:l 00, ~2 A, D D O1 ~ 12. OD D and ~s, Electroelute the band showing o no o and mix it with 5Q mM EDTA. After that, autoradiography is performed under 5DS-PAGE. B is O Tradiodarum: 1st row, material at 8,000 m, W; 2nd row, 12. 000 m, vy, band; 6th column, 24,000 m, w, band; 4th row , s　o, o　o　o　m, w, band. All the bands here are m, w, s , o o o o o moving in one band.

Table 1 shows the amino acid composition of 5IR8-C7.

Table 2 presents a summary of various indeaf purifications of 5IR8.

Table 3 shows 5IR8-C7, SlR8-C6, sxRs-C5 and SlR8- The partial amino acid composition of β7 is shown.

Separation and purification of S lR5-α and S lR8-β For short separation of S R8, 69 6. D2.6 hybridoma cells (1-2 x 106/ml) in serum-free RPM After culturing in 1640 medium for 3 days, the E3IR8-containing supernatant was collected.

The hybridoma cell line 396, D2.6 was as reported (T, M, Aune et al., “Purification and partial character. rization of the lymphokine 5olable engineering mune response 5uppreseor", J, Immunol , Vol. 131, p. 2848 (1983)) and its properties have been analyzed. Book Cell line 10% horse serum (C0 Laboratory θB, New York) were maintained in RPM Engineering 1640-enriched medium containing (Grand Island, State).

The bioassay used to detect S-R8 activity is as reported ( T, M, Aune and W.

Pierce, 1984, 'Mechanism of SlR8acti onat the ce: L’1ular and biochemical 1evel', E.

Lymphokine, Volume 9, edited by Pic, New York, Academ ic Press Publishing, p. 257). The measurements used were mainly tests on sheep red blood cells. This is an intratubular zolag-forming cell response, in which S-R8ox was detected 24 hours before the assay. It inhibits the appearance of IBM or engineered gG antibody-forming cells when added to (T, M , Aune and C.W., Pierce, 1981,' Mechanis m of action of macrophage-derived sup presor factor produced by 5olable im muneresponse 5uppreBsor-treat4macr ophages’, J.

Engineering mmuno 1.127, page 668; T, M, Aune, and C, W, Pierce, 1981, 1st dentification ancL initial character8rization of a non-sp ecificsuppressOr　factor　produced by　 5olable immune response 8uppressor-tu atl noacrophages', J.

Immuno: L, 127 S, 1828 pages; T, M, hunθ and 0, W, Pierce, 1981,'0OnVerSiOn ofso Luble Immune Response 5uppressor to macrophage-derivecL 5uppressor facto r by peroxide “, ProcMath Acad, Sci, U, S, A, vol. 78, p. 5099); the second assay was performed using S engineering R8ox. inhibition of division in 24-hour culture of mast cell tumor cell line P814 (T M, Aune, 1984, 'Modification ofCel lular protein 5ulfhydrul groups by a activated soluble immune response 5upp resor”, J.

(Engineering mmuno volume 1133, page 899). In either case, dilute the 5IR8-containing fraction. The inhibitory ability of 5IR8 is expressed as the inhibitory value (SU). SU is 50% Calculated by the reciprocal of the dilution showing inhibition (T, M, Aune and O, W, P ierce.

1984, 'Modification of ce'1 lular pr oteinsulfhydrul groups b7 activatea 5olable immune response 5uppreasor “, J, Eng. mmuno 1.133, p. 899). To confirm the existence of SlR8 For the biologically active fraction, dicytolidele or is remeasured in the presence of β-mercazotoethanol (T, M, Aune and Pierce, C.W., 1981, 'Mechanism of ac tion Of macrophage - aerivea 5uppress or factor produced b7 soluble immunity response 5uppressor-treatea macropha ges/', J, Engineering mmuno 1.127, page 368; T, M, Aun e and c, w, Pierce, 1981,’ dentificat ionand 1nitial character 1zation of a non-spθC1ficsuppreesor factor prod ucecl by 5olable immune response 5upp rassor-treata macrophages', J.

Immunol, volume 127, page 1828; T, M, Aune and C, W, Pierce, 1981)' Conversion 0f801ub le immunerθ8ponse8uppre8EIOr to macr opha-gaderived 5uppressor factor by peroxide.

Proc, Natl, Acad, Sci, USA, Volume 78, Page 5099 ).

B, high performance liquid chromatography (HPLO) SlR9-α and SlR8-β The separation and purification of C was performed essentially as summarized in FIG.

Ultrafilter 16 liters of serum-free culture supernatant of hybridoma 393, D2.6 cells. Concentrate by filtration and resuspend in 1M pyridine, 0.5M acetic acid - pH 5.5. Chromatograph on Fadex G-50. Collect and recycle the bioactive fractions. Closolve RP-3 or RP-18 column (pore size 60A; 25-40 μm Column size: 9X250), 1M pyridine 10.5M acetate buffer, P Apply to H5.5. Elute with a stepwise gradient of SlR8-n-nolopanol, It is separated into SlR8-α and 5IR8-β. Figure 2 shows the separation into α and β types. Shown below. The fractions containing SlR8-α were collected and treated with Licrosolv RP-18 (pore size 1 00A, 10 μm beads: Column size: 4.2 x 250 mm) column as described above. Rechromatograph using the same buffer and liquid organic phase. In Figure 3 As shown, bioactivity increases from the end of the 20% n-propatur elution to the 30% n-propanol elution. It is eluted as a rather broad peak around the beginning of Ropatool.

The biologically active fraction was then analyzed based on either the 20% late eluting or the 30% early eluting. Then, it was fractionated into S engineering R8-αI and SlR8-α■. SlR8-αI and A wide pore diphenyl column (J, 1M pyridine in T, Bakθr1 (Philinosburg, New Chassis) / Shadow using 0.5 M acetate buffer, PH5.5, straight line of n-propatool Elute using a gradient (Figure 4A) or a step-by-step gradient program (Figure 4B) do. Both SlR6-αI and S-RS-α■ were found in the protein peak region. It eluted. As a result of analyzing the biological activity peak with 5DS-PAi, more than one protein was detected. A band of white matter was found to be present. For this reason, preparative isoelectric focusing (I EF) to separate biological activity from other contaminants.

SlR3-β was also purified and concentrated by reverse phase HPLC (see Figure 1). first S After separation from lR8-α, SlR8-β was chromatographed on Licrosolv RP-18. chromatography on a paycar diphenyl column. It was. As shown in Figure 5A, the bioactive S-engine R8-β was 30% It eluted at , and contained two protein peaks. Filter this material on a diphenyl column. Contrary to chromatography, the biological activity of 30% n-propanol elution is significant for proteins. It coincided with the peak (Figure 5B). In the previous chromatography, there were two SlR8-β Since it eluted between the peaks of I thought maybe he wasn't there. As in the case of SlR9-α, SlR8-β also Furthermore, it was decided to purify the product using preparative EF.

SlR8'i) Purification by IPLC reveals clearly different numbers of 5IR8. The following types were identified: S-R8-αI, SlR8-α■ and SlR9-β.

5DS-PAGR results showed that these bioactive forms were also not pure.

Tablet preparation to increase purity and determine other biochemical parameters (isoelectric point) f: Using SlR8-α■, SlR8-α■ and SlR8-β individually, The isoelectric focusing of the delta was performed using the LKB multiphore system (LKB, Sweden, Zoro). (ma) was used. The pH range of the ampholyte used is 6 to 9. protein is recovered with deionized water from the del: pH' for each fraction and 5IR8. The presence of activity was confirmed. The results are shown in Figure 6. 5) R8-α is approximately pH 6,o It appears as a single peak in , and is hereinafter referred to as 5IR8-α6.

SlR8-α■ appears as two peaks at approximately pl (7 and pH 5); They are called SlR8-α7 and SlR8-α5, respectively.

SlR8-β gathers in a single peak near P) (7), and SlR8-α7S engineering R8- Amino acid analysis of α7 was performed on 0.54μp of natural SIR5-α7 sample. The analysis was carried out using a fluorescamine amino acid analyzer. Amino acid analysis is shown in Table 1. I stopped it.

Table 1 Amino acid mole percent Glx 1 3-00 4f Average value of two analyzes Determination of m, w, isoforms of SlR8 to further analyze the in-type of SlR8 , each &FS engineering RB-α and Hi S engineering R8-β7 with Supercosyl LC knee 18-DB , /75 mm (A-6X 150 tnm; 5upelco engineering nc, pe The protein was desalted by chromatography using The quality was eluted with n-propatool.

SlR8-a isoform has 20% and SlR8-β7 has 30% n-propatrotool. It eluted.

Final purification (removal of ampholytes) and molecular weight determination of SIR8 were carried out in tandem B Lo-511-Tsk-125-T8 with 1250 columns (approximately 7.5 x 300 m m, Bio Rad Laboratories.

(Suchmond, California).

Column is mouse If! ;G (160,000m, w, ), bovine serum albumin (466,000 m, w,), egg albumin (45,000 m, w,), chicken F’//-)y” (24, ODOm, W, ) and lysozyme (14,00 It was prepared with a mixture of 0 m, w, ). The results are shown in Figure 7. All iso of 5IR8 The mold exhibited a molecular weight of approximately 11.0001.

Both SlR8-α and SlR8-β m,w, ~11.00D in HPLO The finding that 'e was shown was previously reported (T, M.

Unlike Aune et al., J. Eng. vol. 01.161, p. 2848 (1983). In this report, HPLC-purified SlR8 (probably SlR8-α) 1ge5D i4. on S-PAGE. oo.

Or 21.000 It has been revealed that it exists as a molecular species of In, W, Ru. Since the previous report, an attempt to detect purified S-R8 on a silver-stained 5DS-PAGE gel has been carried out. I tried this many times, but it didn't match. Investigate the problem of m, w, and the discrepancy in silver staining. In order to avoid It became. Approximately 100 n9 5IR8- After β7k HpLc, freeze-dry to remove the amphoteric electrolyte from the preparation EF.

Re-quench the sample in 10 μl of phosphate buffered saline containing 0.05% SDS. . The sample was then mixed with 10 μl of Ha125 (1,2 mC!i) and Pierce (1! Chemical Co., Rockford, Illinois) ) and incubate the mixture for 10 minutes at room temperature. Sump after 10 minutes Place the sample on a Biodel p-6 column and add the radiolabeled S1R8-β to a phosphate buffered salt. Separate free iodine in solution.

1251-labeled SlR to analyze m, w, and polypeptide composition. 8-β7 f Laemmli's method (ty, K.

Laemmli - 1970, 'Cleavage of 5tructu ralproteins during assembly of the h ead ofbacteriophage T4 ``cleavage of structural proteins during head assembly process'', Nature, vol. 227; p. 680 ) and subjected to 5DS-PAGE using 15% polyacrylamide gel.

Electrophoretic invasion gels are fixed in 50% methanol 15% acetic acid. cellophane wet gel Wrap it in a bag and place it on X-ray film (X-Omat Rochester, York) and subjected to autoradiography (Figure 8). . From the results shown in Figure 8A, most of the proteins move to m, w, ~8000. However, under reducing conditions, bands were observed between m and w between 20,000 and 35,000. Ru. SlR8 is an iron-dependent protein (T, M, Aune and C, W, Pie rce, 1984, 'Mechanism of SlR8action at the cellular and biochemica'l 1eve Lymphokines, edited by E. Pick, 9th Lane, New York. Academic Press, p. 257), and its biological activity was determined by EDTA. is known to be lost; S-Ri9 forms iron-related aggregates. Therefore, the high m, w, SlR8 is similar to the S engineering R8-β. It may be possible to remove it by treatment with EDTA. Therefore, each band is electrolytically melted. The mixture was mixed with 50 μM EDTA and incubated at 4°C overnight. Then lyophilize the sample It was dried, suspended in sample buffer, and subjected to 5DS-PAGE as before. Figure 8B As shown in , this treatment completely eliminates the S-work R8 of high m, w, and therefore the This is suggested to be due to the formation of the iron-8IR8-β7 complex.

A summary of the purification of various in forms of SlR8 is shown in Table 2.

Table 2: Refining table Rough S, N, 27.0xIQ' 1.4x108200 1 Sephadex G-5 07,0x105N, D, -8 engineering R8α N, D, 6.7X1012-8 engineering R Sαl RP-18N, D, Kashino 1010-8 Engineering R8α5 Engineering EF N, D, 3xICl Yu1 - -8IR8α6EF N, D, 1xlO1O--8 Engineering R8α7IEF N, D, 2X1010-'5HjSa5 Supercosil < 1.0 2.8x1012>2.8x1012>1.4XlO”5IR8a 6. z, Percosi pv <I Jl 1.8X1012>1.3x1012>9 ．． 0x101O8 Engineering R8α7 Supercosyl <1.0 2.8x1011>2. 8x1011>1.4x109S engineering R8a5 'rSx <i, 0 1x10 11　＞1.0Xi011　＞5XiO”S Engineering R8(!6TSK　＜1.0　1 xID">1.DXloll>5X10"S engineering R8Iα7TSK <1.0 1x1011>1.0xlO11>5xlO"S engineering R8β N, D, 7.7X ICl Yu〇-8 Engineering R8βRP-18N, D, 4X101O--8IR8β Schiff: r-Nil 783 4xlO” 5.1XIO’ 2.6X10”5IR 8β7E EF N, D, 2x10”--8E R8β7 Supercosyl 1. 0 2×1010〉2.0×1010〉1.0×108S work R8/7TSK <1.0 7x1011>7.0x1011>3.5x10'1, rough adjustment The protein of the product was measured by the Lowry method (12).

Protein analysis on samples from HPLC was performed using fluorescamine. (1D).

2. The initial capacity is 16 liters Example 5 Mouse samples were prepared as described in Examples 1-4.

Oval W = product was hydrolyzed using 6'MHC6 and fluorescamine detection method was used Partial amino acid analysis was performed according to conventional methods. ateln et al., 1976, A. rch.

See Biochem, Biophys, Volume 155; Pages 203-212. and. Table 3 shows the amino acid composition of the protein in mole percent.

Table 3 Partial amino acid analysis of S engineering R8 5IR8-a7+ S-work R8-a6” S-work R8-α5” S-work R8-β70A SX 8.1 11.6 12.3 10.0Thr A, 6 6.8 5.6 3.7Ser 5.5 8.0 6.3 14.8GIX 12.5 12. 6 17.2 13.8aly 12.0 1C1,79,6'I 7. dAl a 9.6 2.5 7.5 5.5 Val 7.0 3.9 6-7 8.6 Leu s, i 9.5 9.0 5.9Tyr 2.7 5.0 2.3 3 ．． 8Phe 4.3 n, d 3.6 3. IHLs 2.4 3.0 1. 8 2.2L7E! 4.8 6.5 8.5 3.7Arg 4.7 1.2 2.9 3.3 Case 6 Mouse knee cell hybridoma 393. S engineering R9-α7 protein obtained from D2.6 The partial amine terminal sequence of white matter was determined by the following method.

According to the purification procedure of Example 1-4, S engineering R8-α7 protein was DFAE-chromatographed. E, isolated by reversed-phase high performance chromatography and isoelectric focusing. 10 0 pmol S engineering R8-a7 f Applied Systems vapor phase sea It was subjected to automated sequence analysis using a sequencer and an automated program. PTH amino Acid analysis identified 21 amino acids from the amine end. Partial amine terminal The columns are shown below.

Met Thr Glu Glu Asp Gln Gln Ser Ser Gln Pro Lys13 1, ii 15 16 17 18 19 20 21Thr Thr Engineering Asp Asp Ala Gly Asp 5e rFIGIIRE 1 ↓ Tfu? Day72G-50 ↓ Riku O Tsurua RP-8) IPLc 20Z F00 no ψ nor 1 30z Flo Q no ψ nor ↓ ↓ 5IR5-a 5IRS-B FIGLIRE 2 i thing shi 3t + i (S single i fL) E king] n-flo 0 no ψ nor 5 et al. --- F! GtI;Iε3 Biological Divination I・King (S Takudo IiL) Mouth n-fu 0〇 to O Nor 5 et al 111- FIGLIRε4 FiGLi;IE 5 Eirei (rei) FIGuRE 6 FIGIIREA ■ L? international search report lmemma″al Ao61′callas N6■,, 9s8610222 61ms+nalIInsl^”””””Pc〒/rJs86102226

Claims (9)

[Claims] 1. Approximately 11,000 m.p. by molecular sieve chromatography. w. and SR Assays by in vitro antibody-forming cell responses to BC A soluble immune response inhibitor with a specific activity of 1.0 x 1011-7.0 x 1011 SU. isoforms of antiproteins. 2. The soluble immune response according to claim 1, which is SIRS-α5 having the following properties: Answer inhibitory protein isoforms. (a) μg of protein as assayed by in vitro antibody-forming cellular responses to SRBC. Specific activity of 1.0 x 1011 SU or more per substance, (b) Migrating as a single substance at approximately pH 5.0 in isoelectric focusing for the preparation of granular gels. move, (c) have a partial amino acid composition contained in the following table. Asx 12.3 Thr　5.6 Ser　6.3 Glx 17.2 Gly　9.6 Ala 7.5 Val　6.7 Met　1.3 Ile 5.3 Leu　9.0 Tyr　2.5 Phe 3.6 His 1.8 Lys 8.5 Arg　2.9 3. The soluble immune response according to claim 1, which is SIRS-α6 having the following properties: Answer inhibitory protein isoforms. (a) μg of protein as assayed by in vitro antibody-forming cellular responses to SRBC. Specific activity of 1.0 x 1011 SU or more per substance, (b) Migrating as a single substance at approximately pH 6.0 in isoelectric focusing for the preparation of granular gels. move, (c) have a partial amino acid composition contained in the following table. Asx 11.6 Thr　6.8 Ser　8.0 Glx 12.6 Gly 10.7 Ala 2.5 Val　5.9 Met　1.0 Ile　3.2 Leu　9.5 Tyr 5.0 His 3.0 Lys 6.5 Arg　1.2 4. The soluble immune response according to claim 1, which is SIRS-α7 having the following properties: Answer inhibitory protein isoforms. (a) μg of protein as assayed by in vitro antibody-forming cellular responses to SRBC. Specific activity of 1.0 x 1011 SU or more per substance, (b) Migrating as a single substance at approximately pH 6.0 in isoelectric focusing for the preparation of granular gels. move, (c) have a partial amino acid composition contained in the following table. Asp 8.45 Thr 4.66 Ser　　5.51 Glx 13.00 Gly 11.97 Ala 11.95 Val 8.02 Met 1.87 Ileu　5.69 Leu 9.20 Tyr 2.77 Phe 4.28 His 2.33 Lys 4.72 Arg 5.64 5. The soluble immune response according to claim 1, which is SIRS-α7 having the following properties: Answer inhibitory protein isoforms. (a) μg of protein as assayed by in vitro antibody-forming cellular responses to SRBC. Specific activity of 1.0 x 1011 SU or more per substance, (b) In isoelectric focusing for the preparation of granular glue, it is migrated as a single substance at approximately pH 7.0. move, (c) Has a partial amino acid composition included in the following table, Asx 8.1 Thr　4.6 Ser　5.5 Glx 12.5 Gly 12.0 Ala　9.6 Val　7.0 Met　1.8 Ile 5.0 Leu　8.1 Tyr　2.7 Phe 4.3 His 2.4 Lys 4.8 Arg　4.7 (d) The amino terminal sequence is [There is an array]. 6. The soluble immune response according to claim 1, which is SIRS-β7 having the following properties: Answer inhibitory protein isoforms. (a) μg of protein as assayed by in vitro antibody-forming cellular responses to SRBC. Specific activity of 7.0 x 1011 SU or more per quality, (b) Migrating as a single substance at approximately pH 7.0 in isoelectric focusing for the preparation of granular gels. move, (c) have a partial amino acid composition contained in the following table. Asx 10.0 Thr　3.7 Ser　14.8 Glx 13.8 Gly　17.4 Ala 5.5 Val　8.6 Met　0.9 Ile 3.3 Leu　5.9 Tyr　3.8 Phe 3.1 His　2.2 Lys 3.7 Arg　3.3 7. Soluble according to claim 1, suitable for parenteral administration for the prevention of organ rejection An effective amount of at least one isoform of an immune response suppressing protein and a conventional drug A pharmaceutical preparation consisting of an oral carrier substance. 8. Antibodies formed against the isoform of the soluble immune response suppressing substance according to claim 1 . 9. The antibody according to claim 8, which is a monoclonal antibody.