EP0245432A1

EP0245432A1 - Isoforms of soluble immune response suppressor

Info

Publication number: EP0245432A1
Application number: EP86907042A
Authority: EP
Inventors: Carl W. Pierce; David R. Webb, Jr.
Original assignee: F Hoffmann La Roche AG; Jewish Hospital
Current assignee: F Hoffmann La Roche AG; Jewish Hospital
Priority date: 1985-10-22
Filing date: 1986-10-21
Publication date: 1987-11-19
Also published as: JPS63501572A; WO1987002677A1; EP0245432A4

Abstract

En utilisant la chromatographie liquide de haute performance et la focalisation isoélectrique, on a isolé quatre isoformes différentes, biologiquement actives de protéines immunodépressives (SIRS-alpha5, SIRSalpha6, SIRS-alpha7 et SIRS-beta7) (Fig. 1). Lorsqu'elles sont soumises à la chromatographie à travers un tamis moléculaire, toutes ces isoformes ont pratiquement le même poids moléculaire, 11 000 environ.Using high performance liquid chromatography and isoelectric focusing, four different, biologically active isoforms of immunosuppressive proteins (SIRS-alpha5, SIRSalpha6, SIRS-alpha7 and SIRS-beta7) were isolated (Fig. 1). When subjected to chromatography through a molecular sieve, all of these isoforms have substantially the same molecular weight, approximately 11,000.

Description

ISOFORMS OF SOLUBLE IMMUNE RESPONSE SUPPRESSOR

Summary of the Invention

The present invention relates to novel isoforms of soluble immune response suppressor protein. By using high performance liquid chromatography and isoelectric focusing four different biologically active isoforms were isolated. iσ

The isoforms of the present invention are useful for prophylaxis of organ rejection and for suppression of cell mediated reactions.

15 Background of the Invention

Soluble immune response suppressor (SIRS) is an antigen-nonspecific suppressor factor produced by Ly-2 T cells activated with concanavalin A or interferon (Aune, T.

20 M., and C. W. Pierce, 1984, "Mechanism of SIRS action at the cellular and biochemical level", in Lymphokines, Vol. 9, edited by E. Pick, Academic Press, New York, p. 257). It inhibits immune responses and blocks cell division in a variety of normal and transformed cell lines (Aune, T. M.,

25 and C. W. Pierce, 1981, "Mechanism of action of macrophage- derived suppressor factor produced by soluble immune response suppressor-treated macrophages", J. Immunol. 127:368; Aune, T. M., and C. . Pierce, 1981, "Identification and initial characterization of a non-specific suppressor factor produced

30 by soluble immune response suppressor-treated macrophages", J. Immunol. 127:1828; Aune, T. M., and C. W. Pierce, 1981, "Conversion of soluble immune response suppressor to macrophage-derived suppressor factor by peroxide", Proc. Natl. Acad. Sci. USA 78:5099). Recent studies by Aune and

35 Pierce (Aune, T. M., and C. . Pierce, 1981, "Conversion of soluble immune response suppressor to macrophage-derived suppressor factor by peroxide", Proc. Natl. Acad. Sci. USA

78:5099) established that SIRS must be activated by H-0-

(SIRSox) to exert its biolog^aic effects. Further, SIRS action is blocked by sulfhydryl reagents, catalase, p-aminobenzoic acid, ascorbic acid, taurine, or potassium iodide; SIRS UΛ appears to mediate inhibition of cell division by causing oxidation of cellular protein sulfhydryl groups (Aune, T. M., 1984, "Modification of cellular protein sulfhydryl groups by activated soluble immune response suppressor", J. Immunol.

133:899). SIRS also disrupts the normal array of intracellular microtubules visualized by immunofluorescent microscopy and inhibits assembly of purified microtubule proteins in cellfree systems (Irons, R. D. et al., 1984, "Soluble immune response suppressor (SIRS) inhibits microtubule function in vivo and microtubule assembly in vitro", J. Immunol. 133:2032). Intracellular integrity of microtubules as well as GTP-dependent assembly of microtubule proteins are known to be sensitive to sulfhydryl oxidizing agents.

Analysis of the biochemical structure of the SIRS pro ein(s), has also been undertaken (Aune, T. M., Webb, 1983, "Purification and partial characterization of the lymphokine soluble immune response suppressor", J. Immunol. 131:2848? and Aune, T. M. et al., 1983, "Purification of radiolabeled soluble immune response suppressor", in Interleukins, Lymphokines and Cytokines, edited by J. J. Oppenheim and S. Cohen, Academic Press, New York, p. 383). The initial studies showed that two peaks of biologic activity could be recovered after reverse-phase high performance chromatography. One peak eluted with 20% propanol (hereinafter referred to as SIRS-α) and the second peak eluted with 30% propanol (hereinafter referred to as SIRS- ) (Aune, T. M., 1983, "Purification and partial characterization of the lymphokine soluble immune response suppressor", J. Immunol. 131:2848). SIRS-α was subsequently purified extensively by using C-18 and diphenyl-modified silica supports for reverse-phase liquid chromatography

(Aune, T. M., 1983, "Purification and partial 5 characterization of the lymphokine soluble immune response suppressor", J. Immunol. 131:2848). After chromatography on a diphenyl column, two peaks of bioactivity were observed.

When subjected to SDS-polyacrylamide gel electrophoresis

(SDS-PAGE), the first peak was found to contain a single

10. detectable protein with a m.w. of 14,000, and the second peak contained a single protein with a m.w. of 21,000. In that report no attempt was made to characterize SIRS-6.

Experiments were also carried out by using SIRS made in

15 a cellfree mRNA translation system (Nowowiejski-Wieder, I. et al., 1984, "Cellfree translation of the lymphokine soluble immune response suppressor (SIRS) and characterization of its

+ mRNA", J. Immunol. 132:556). Poly A RNA was isolated from

393D2.6 hybridoma cells, fractionated by methyl-mercury

20 agarose gel electrophoresis, and translated by using a rabbit reticulocyte lysate translation system. RNA coding for SIRS activity was recovered in two fractions: one corresponded to a size of 25S and produced SIRS with m.w. of 14,000 and 8000, and the second corresponded to a size of 21 to 22S and

25 produced SIRS with a m.w. of 8000. This established for the first time the possibility of an underlying genetic basis for the existence of more than one form of SIRS.

Detailed Description of the Invention

30

The present invention relates to novel isoforms of soluble immune response suppressor (SIRS) (SIRS-α5, SIRS-α6, SIRS-α7, and SIRS-37) having a molecular weight (m.w.) of

35 about 11000 on molecular sieve chromatography and having a specific activity of from about 1.0 x 10 - 7.O x 10 Units of suppression (SU)/μg of protein when assayed by in vitro antibody-forming cell response to sheep erythrocytes (SRBC).

For details of bioassays used to detect SIRS activity see example 1.

The novel isoforms of SIRS protein of the present invention can be obtained by utilizing one or more high pressure liquid chromatography (HPLC) steps followed by preparative isoelectric focusing (IEF). In the preferred embodiment of the present invention the novel isoforms of SIRS protein can be obtained as outlined in Fig. 1.

The novel isoforms of SIRS protein of the present invention obtained by the aforesaid processes each focused as a single peak of SIRS activity on preparative IEF in a granulated gel (for details see example 2). The specific activity of the isoforms of SIRS protein was found to be in the range of 1.0 x 10 - 7.0 x 10 SU/μg of protein when assayed by in vitro antibody-forming cell response to SRBC (for details see Table 2). Analysis of the various isoforms of SIRS protein suggested that all forms have a roughly equivalent m.w. of about 11000.

Furthermore, it was found that the preferred isoform of SIRS protein of the present invention, i.e. SIRS-α7, has a partial amino acid composition as contained in the following table:

Asp : 8.45

Thr : 4.66

Ser : 5.54

Glx • 13.00 Gly 11.97

Ala : 11.93

Val 8.02

Met ^■ 1.87 lieu : 5.69

Leu 9.20

Tyr 2.77

Phe 4.28

His 2.33

Lys 4.72

Arg : 5.64

The novel isoforms of SIRS protein of the present invention can be used for the prophylaxis of organ rejection, e.g. in kidney, skin, heart, pancreas, bone marrow, small intestine and lung allogenic transplants.

The individual isoforms of SIRS protein can be utilized per se or alternatively mixtures of two or more of such isoforms can be employed. Such mixtures can be obtained by mixing the isolated isoforms as derived or stopping the purification procedure where several isoforms of SIRS protein are present but no non-SIRS active proteins are present so that the composition is a mixture of isoforms of SIRS protein.

The novel isoforms of SIRS protein of the present invention may be combined with conventional pharmaceutical parenteral carrier material to provide pharmaceutical preparations suitable for prophylaxis of organ rejection.

The novel isoforms of SIRS protein may be administered by parenteral application either intravenously, subcutaneously, intra-muscularly or orally. The dosage of these compounds is dependent upon various factors such as the particular compound employed and the particular formulation.

The dosage may parallel that currently being used for prophylaxis of organ rejection.

Furthermore, antibodies in particular, monoclonal antibodies can be raised against the novel isoforms of SIRS . . protein according to the present invention in a manner known per se. These antibodies can be used in a well known manner for diagnostic or therapeutic purpose as well as for purification purposes.

The present invention will be better understood on the basis of the following examples when considered in connection with the following figures and tables:

Figure 1 shows a flow diagram of SIRS purification and resolution of the isoforms.

Figure 2 shows the separation of SIRS-α and SIRS- by reverse-phase HPLC. Bioactive fractions from a Sephadex G-50 column were applied to a Lichrosorb RP-18 column. The flow rate used was 2 ml/min; 4-min fractions were collected, and 1% of each fraction was assayed for biologic activity.

Figure 3 shows further purification of SIRS-α by reverse-phase HPLC on Lichrosorb RP-18. SIRS-α, which eluted in 20% propanol from the initial column, was reapplied to a reverse-phase Lichrosorb RP-18 column. The flow rate was 0.5 ml/min; 4-min fractions were collected; 1% of each fraction was assayed for SIRS activity.

Figure 4 shows chromatography of SIRS-αl and SIRS-αll on a Bakerbond diphenyl reverse-phase column. SIRS-αl and SIRS-αll were separately run on a Bakerbond diphenyl column at a flow rate of 0.5 ml/min; 2-min fractions were collected 7 and 1% of each fraction was assayed for SIRS activity . A, SIRS- αl ; B , SIRS- αl l . In A, prote in was eluted with a l inear 0 to 60% n-propanol grad ient ; in B , prote in was eluted wi th a stepwise n-propanol grad ient .

Figure 5 shows further chroma tography of SIRS- on

Lichrosorb RP-18 (A) and Bakerbond diphenyl (B) reverse-phase columns. SIRS-3, which eluted in 30% propanol on Lichrosorb

RP-18, was rechromatographed on Lichrosorb RP-18 at a flow rate of 0.5 ml/min. The biologically active fractions were pooled and subjected to chromatography on a Bakerbond diphenyl column. In each case 1% of each fraction was analyzed for bioactivity.

Figure 6 shows resolution of SIRS-α and SIRS- by IEF. The pooled, bioactive fractions of SIRS-αl, SIRS-αll, and SIRS-3 were separately subjected to IEF in a granulated gel. The gel was eluted with deionized water. After assessing the pH of each fraction, 1-μl samples were assayed for SIRS activity.

Figure 7 shows molecular sieve chromatography of the SIRS isoforms. SIRS purified by reverse-phase HPLC and IEF was subjected to high-performance molecular sieve chromatography. Each fraction was assayed for SIRS activity. Arrows mark the elution time of the protein standards: mouse IgG (160,000); BSA (66,000); ovalbumin (45,000); trypsinogen (24,000); and lysozy e (14,000).

Figure 8 shows SDS-PAGE of SIRS-37 radiolabelled with

1 "j e

I. A, an I SIRS-37 autoradiogram after SDS-PAGE under reducing conditions. The most prominent band has a m.w. of/ 8000. The bands shown at m.w. , vl2,000 , and 8000 were electroeluted from the gel, mixed with 50 iaM EDTA, and subjected to SDS-PAGE and autoradiography. B, The autoradiogram: lane 1, 8000 m.w. material; lane 2, .12,000 m.w. band; lane 3, 24,000 m.w. band; lane 4, 30,000 m.w. band. All bands now migrate at a single m.w. of 8000.

Table 1 presents the amino acid composition of SIRS-α7,

Table 2 presents a summary of the purification of the various isoforms of SIRS.

Table 3 presents the partial amino acid compositions of SIRS-α7, SIRS-α6, SIRS-α5 and SIRS- 7.

Example 1

Separation and Purification of SIRS-α and SIRS- A. SIRS production and bioassay

For the isolation of SIRS 393.D2.6 hybridoma cells (1 to 2 x 10 /ml) were cultured in RPMI 1640 medium without serum supplement and for 3 days before harvesting SIRS-containing supernatant fluid.

The hybridoma cell line 393.D2.6 was constructed and characterized as described (Aune, T. M. et al., "Purification and partial characterization of the lymphokine soluble immune response suppressor", J. Immunol. 131, 2848 [1983] ). It had been maintained in supplemented RPMI 1640 medium containing 10% horse serum (GIBCO Laboratories, Grand Island, NY).

The bioassays used to detect SIRS activity have been described (Aune, T. M., and C. W. Pierce, 1984, "Mechanism of SIRS action at the cellular and biochemical level", in Lymphokines, Vol. 9, edited by E. Pick, Academic Press, New York, p. 257). The assays used were the primary in vitro plaque-forming cell response to sheep erythrocytes, in which SIRS inhibits the appearance of IgM or IgG antibody-forming /__. cells when added 24 hr before assay (Aune, T. M., and C. W.

Pierce, 1981, "Mechanism of action of macrophage-derived suppressor factor produced by soluble immune response suppressor-treated macrophages", J. Immunol. 127:368; Aune,

T. M., and C. W. Pierce, 1981, "Identification and initial characterization of a non-specific suppressor factor produced by soluble immune response suppressor-treated macrophages",

J. Immunol. 127:1828; Aune, T. M., and C. W. Pierce, 1981,

"Conversion of soluble immune response suppressor to macrophage-derived suppressor factor by peroxide", Proc.

Natl. Acad. Sci. USA 78:5099); the second assay was the inhibition of division of the Mastocytoma cell line P815 by

SIRS in a 24-hr culture (Aune, T. M., 1984, "Modification ox of cellular protein sulfhydryl groups by activated soluble immune response suppressor", J. Immunol. 133:899). In all cases SIRS-containing fractions were subjected to dilution analysis and the suppressive capacity of SIRS was reported in units of suppression (SU). The SU were calculated as the reciprocal of the dilution that gives 50% suppression (Aune, T. M., and C. W. Pierce, 1984, "Modification of cellular protein sulfhydryl groups by activated soluble immune response suppressor", J. Immunol. 133:899). To authenticate the presence of SIRS bioactive fractions were routinely reassayed in the presence of dithiothreitol or 3- mercaptoethanol to inhibit SIRS activity (Aune, T. M., and C. W. Pierce, 1981, "Mechanism of action of macrophage- derived suppressor factor produced by soluble immune response suppressor-treated macrophages", J. Immunol. 127:368; Aune, T. M., and C. W. Pierce, 1981, "Identification and initial characterization of a non-specific suppressor factor produced by soluble immune response suppressor-treated macrophages", J. Immunol. 127:1828; Aune, T. M., and C. W. Pierce, 1981, "Conversion of soluble immune response suppressor to macrophage-derived suppressor factor by peroxide", Proc.

Natl. Acad. Sci. USA 78:5099). B. High performance liquid chromatography (HPLC)

Separation and purification of SIRS-α and SIRS- was done essentially as outlined in Fig. 1.

16 liters of serum-free culture supernatant fluid from hybridoma 393.D2.6 cells were concentrated by ultra- filtration, resuspended in 1M pyridine, 0.5M acetic acid, pH 5.5 and chromatographed on Sephadex G-50. The bioactive fractions were pooled and applied to a Lichrosorb RP-8 or RP-18 COliimiT ( 60A pore size; 25- to 40-μm bead; column dimensions;-* 9 x 250 mm) in a 1M pyridine/0.5M acetic acid buffer, pH 5.5. SIRS was eluted by using a stepwise gradient of n-propanol and separated into SIRS-α and SIRS- . Separation into the α and 3 forms is shown in Fig. 2. The fractions containing SIRS-α were pooled and rechromatographed on a Lichrosorb RP-18 resin (100A pore size; 10 μm bead; column dimensions: 4.2 x 250 mm) by using the same buffers and fluid organic phase as described above. As seen in Figure 3, the bioactivity eluted in a rather broad peak near the end of the 20% n-propanol step and in the beginning of the 30% n-propanol step.

The bioactive fractions were then divided into SIRS-αl and SIRS-αll on the basis of whether they eluted in the later

20% step or early 30% step. SIRS-αl and SIRS-αll were applied separately to a Bakerbond, wide-pore diphenyl column

(J. T. Baker, Phillipsburg, N.J.) by using a 1M pyridine/0.5

M acetic acid buffer, pH 5.5 and eluted with n-propanol by using either a linear gradient (Fig. 4A) or a step gradient program (Fig. 4B); both SIRS-αl and SIRS-αll elute in a region containing protein peaks. Analysis of these peaks of bioactivity by SDS-PAGE revealed the presence of more than one band of protein. For this reason, it was decided to separate the bioactivity from remaining contaminants by preparative isoelectric focusing (IEF). SIRS-3 was also purified and concentrated by reverse- phase HPLC (See Fig. 1). After the initial separation from SIRS-α, SIRS-3 was subjected to chromatography on Lichrosorb RP-18, followed by chromatography on a Bakerbond diphenyl resin. As shown in Figure 5A, the bioactive SIRS-3 eluted in 30% n-propanol and included two protein peaks. This material was chromatographed on a diphenyl column, and the bioactivity eluted in 30% n-propanol coincident with a large protein peak (Fig. 5B). Because SIRS- eluted between two peaks in the preceding chromatography, we were concerned that the diphenyl column had not resolved these components. As was the case with SIRS-α,. it was decided to purify SIRS- further by preparative IEF.

Example 2

Resolution of the isoforms of SIRS-α and -3 by preparative IEF in a granulated gel

The purification of SIRS by HPLC allowed the identification of several, apparently different forms of SIRS: SIRS-αl, SIRS-αll, and SIRS-6. SDS-PAGE had indicated that these bioactive forms were not pure. Preparative IEF was used to increase purity and determine another biochemical parameter ( isoelectric point), SIRS-αl, SIRS-αll, and SIRS-3 were individually subjected to isoelectric focusing in a granulated gel by using an LKB Multiphor system (LKB, Bromma, Sweden). The pH range of the ampholines used was 3 to 9. The protein was recovered from the gel bed by elution with deionized water; each fraction was analyzed for pH, and the presence of SIRS activity was assessed. The results are presented in Figure 6. SIRS-αl focused as a single entity at about pH 6.0 and will hereafter be referred to as SIRS-α6; SIRS-αll focused as two entities at approximately pH 7 and pH 5 and will now be called SIRS-α7 and SIRS-α5, respectively; and lastly, SIRS-3 focused as a single entity near pH 7 and will be referred to as SIRS- 7.

Example 3 Amino acid

Amino acid analysis of SIRS-α7 was performed with the fluorescamine amino acid analyser on 0.54 μg samples of na¬ tive SIRS-α7. Amino acid analyses are summarized in Table 1

Table 1

*

Amino Acid^' Composition (Partial) SIRS-α7

Amino Acid Mol p

Asp 8.45

Thr 4.66

Ser 5.51

Glx 13.00

Gly 11.97

Ala 11.93

Val 8.02

Met 1.87 lieu 5.69

Leu 9.20

Tyr 2.77

Phe 4.28

His 2.33

Lys 4.72

Arg 5.64

* average of 2 determinations Example 4

Determination of the m.w. of the SIRS isoforms by molecular sieve chromatography and SDS-PAGE

To further analyze isoforms of SIRS, the various SIRS-α and SIRS- 7 were desalted by chromatography by using a supelcosil LC-18-DB column (4.6 x 150 mm; Supelco Inc., Bellefonte, PA); protein was eluted with n-propanol. The SIRS-α forms eluted in 20% and SIRS-37 in 30% n-propanol.

Final purification (removal of ampholines) and sizing of SIRS was performed by using tandemly linked Bio-Sil-TsK-125- TsK-250 columns (7.5 x 300 mm each; Bio-Rad Laboratories, Richmond, CA). The columns were calibrated with a mixture of mouse IgG (160,000 m.w.), bovine serum albumin (66,000 m.w.), ovalbumin (45,000 m.w.), trypsinogen (24,000 m.w.) and lysozyme (14,000 m.w.). The results are represented in Fig. 7. All of the isoforms of SIRS exhibit a m.w. of approximately 11,000.

The observation that both SIRS-α and SIRS- displayed m.w. of ^11,000 on HPLC was not consistent with an earlier report (Aune, T. M. et al., J. Immunol. 131,2848 [1983]) in which HPLC-purified SIRS (probably SIRS-α) was found to exist as 14,000 or 21,000 m.w. species on SDS-PAGE. Since the earlier report, repeated attempts to detect purified SIRS by silver staining SDS-PAGE gels have been inconsistent. To examine the question of m.w. and to avoid the inconsistent silver staining problem, SIRS-S7 was lodinated by using 125I as described below. For radioiodination of SIRS- 7, approximately 100 ng of SIRS-37 were lyophilized after HPLC to remove the ampholines from preparative IEF. The sample was resuspended in 10 μl of phosphate-buffered saline containing 0.05% SDS. The sample was then mixed with 10μl Na 125I (1.2mCi), one Iodo-bead (Pierce Chemical Co., Rockford, IL) was added, and the mixture incubated at room temperature for 10 minutes. After 10 minutes, the sample was added to a Biogel P-6 column, and the radiolabeled SIRS- separated from the free iodine in phosphate-buffered saline.

To analyze its m.w. and polypeptide complexity, 125I- labelled SIRS-87 was subjected to SDS-PAGE according to the method of Laemmli (Laemmli, U.K., 1970, "Cleavage of structural proteins during the assembly of the head of bacteriophage T4", Nature 227:680) by using a 15% polyacryl- amide gel. After electrophoresis the gel was fixed in 50% methanol/5% acetic acid. The wet gel was then wrapped in cellophane and placed against x-ray film (X-Omat XAR-5,

Kodak, Rochester, NY) for autoradiography (Fig. 8). The results prescribed in Fig. 8A show that the majority of the protein migrated with an m.w. of/»*8000, but higher m.w. bonds between 20000 to 35000 could be observed under reducing conditions. SIRS is an iron-dependent protein (Aune, T.M., and C. W. Pierce, 1984, "Mechanism of SIRS action at the cellular and biochemical level", in Lymphokines, Vol. 9, edited by E. Pick, Academic Press, New York, p. 257), and it is known that EDTA will destroy its biologic activity; it was reasoned that SIRS might be forming aggregates that involved iron, and thus the higher m.w. forms of SIRS might simply be eliminated by treating SIRS-3 with EDTA. Therefore, each of the bands was electroeluted and mixed with 50μM EDTA and allowed to stand overnight at 4°C. The samples were then lyophilized, resuspended in sample buffer, and subjected to

SDS-PAGE as before. As shown in Figure 8B, this treatment completely abolishes any higher m.w. forms of SIRS, and thus suggests that aggregation may be due to the formation of iron-SIRS-37 complexes.

A summary of the purification of the various isoforms of SIRS is presented in Table 2. Table 2 Purification Table

Total Protein Total Specific Act. Purification

Step μg SU SU/μg Factor

Crude S.N.' 7.0 x 10° 1.4 x 10 200

Sephadex G-50 7.0 x 10 N.D.

SIRSα N.D. 6 .7 X 10¹² -_*

SIRSαl RP-18 N.D. 6 X 10¹⁰ -

SIRSαl diphenyl 105.1 3 X 10¹⁰ 2.9 X 10⁸ 1.5 X IO⁶

SIRSαll diphenyl 446.2 9 X 10¹³ 2.0 X 10¹¹ 1 X IO⁹

SIRSα5 IEF N.D. 3 X 10¹¹ - -

SIRSαδ IEF N.D. 1 X 10¹⁰ - -

SIRSα7 IEF N.D. 2 X 10¹⁰ - -

SIRSα5 supelcosil <α.o 2 .8 X 10¹² >2.8 X 10¹² >1.4 X IO¹⁰

SIRSαδ supelcosil <1.0 1 .8 X 10¹² >1.8 X 10¹² >9.0 X IO¹⁰

SIRSα7 supelcosil <1.0 2 .8 X 10¹¹ >2.8 X 10¹¹ >1.4 X IO⁹

SIRSα5 TSK <1.0 1 X 10¹¹ >1.0 X io¹¹ >5 X IO⁸

SIRSα6 TSK <1.0 1 X 10¹¹ >1.0 X 10¹¹ >5 X IO⁸

SIRSα7 TSK <1.0 1 X 10¹¹ >1.0 X 10¹¹ >5 X io⁸

SIRS3 N.D. 7. .7 X 10¹⁰ -

SIRS3 RP-18 N.D. 4 X 10¹⁰ - -

5

SIRS3 diphenyl 783 4 X 10¹⁰ 5.1 X lϋ⁷ 2.6 X IO³

SIRS37 IEF N.D. 2 X 10¹⁰ - -

SIRSS7 supelcosil <1.0 2 X 10¹⁰ >2.0 X 10¹⁰ >1.0 X IO⁸

SIRS87 TSK <1.0 7 X 10¹¹ >7.0 X io¹¹ >3.5 X io⁹

1 Protein in crude preparations was determined by the method of Lowry (12). Protein determinations performed on samples from HPLC were done using fluorescamine (10). Starting volume was 16 liters. Example 5 Partial amino acid analysis (composition) of mouse SIRS-α7, SIRS-α6, SIRS-α5 and SIRS-37 protein obtained from the T-cell hybridoma 393.D2.6

Mouse SIRS was purified as described in Examples 1-4. The purified material was hydrolyzed using 6 M HC1 and subjected to partial amino acid analysis according to conventional procedures using a fluorescamine detection system. See Stein et al. , 1973, Arch. Biochem. Biophys., 155:203-212. The mole percent of the amino acid composition of the protein are shown in Table 3.

Table 3. Partial Amino Acid Analysis of SIRS Mole Percent

SIRS-α7* S SIIRRSS--α6⁺ SIRS-α5⁺ SIRS-87⁺

Asx 8.1 1 111..6 12.3 10.0

Thr 4.6 6 6..8 5.6 3.7

Ser 5.5 8 8..0 6.3 14.8

Glx 12.5 1 122..6 17.2 13.8

Gly 12.0 1 100..7 9.6 17.4

Ala 9.6 2 2..5 7.5 5.5

Val 7.0 3 3..9 6.7 8.6

Met 1.8 1 1..0 1.3 0.9

He 5.0 3 3..2 5.3 3.3

Leu 8.1 9 9..5 9.0 5.9

Tyr 2.7 5 5..0 2.3 3.8

Phe 4.3 nn..d 3.6 3.1

His 2.4 3 3..0 1.8 2.2

Lys 4.8 6 6..5 8.5 3.7

Arg 4.7 1 1..2 2.9 3.3

* Average of 3 determinations + One determination n.d. not determined Example 6 Determination of the partial amino-terminal sequence of SIRS-α7 protein

The partial amino-terminal sequence of the SIRS-α7 protein obtained from the mouse T-cell hybridoma 393.D2.6 was obtained according to the following procedure:

Following the purification procedure of Examples 1-4 SIRS-α7 protein was isolated by DEAE-chromatography, reverse-phase high performance chromatography and isoelectric focusing. 100 p mole of SIRS-α7 was subjected to automated sequence analysis using the Applied Biosystems gas phase sequenator and automated programs. Twenty-one amino acids were identified from the amino terminus by PTH amino acid analysis. The partial amino-terminal sequence is presented below:

1 2 3 4 5 6 7 8 9 10 11 12

Met Thr Glu Glu Asp Gin Gin Ser Ser Gin Pro Lys

13 14 15 16 17 18 19 20 21

Thr Thr He Asp Asp Ala Gly Asp Ser

Claims

We claim :

1. An isoform of soluble immune response suppressor protein having a m.w. of about 11000 on molecular sieve chromatography and having a specific activity of from about 1.0 x 10 - 7.O x 10 SU/μg of protein when assayed by in vitro antibody-forming cell response to SRBC.

2. An isoform of soluble immune response suppressor protein according to claim 1 which is SIRS-α5 characterized by

(a) a specific activity of >1.0 x 10 SU/μg of protein when assayed by in vitro antibody-forming cell response to SRBC;

(b) focusing as a single entity at about pH 5.0 on preparative isoelectric focusing in a granulated gel; and

(c) a partial amino acid composition as 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

He 5.3

Leu 9.0

Tyr 2.3

Phe 3.6

His 1.8

Lys 8.5

Arg 2.9

3. An isoform of soluble immune response suppressor protein according to claim 1 which is SIRS-α6 characterized by (a) a specific activity of >1.0 x 10 SU/μg of protein when assayed by in vitro antibody-forming cell response to SRBC;

(b) focusing as a single entity at about pH 6.0 on preparative isoelectric focusing in a granulated gel; and

(c) a partial amino acid composition as contained in the following table:

Asx 11.6

Thr 6.8

Ser 8.0

Glx 12.6

Gly 10.7

Ala 2.5

Val 3.9

Met 1.0

He 3.2

Leu 9.5

Tyr 5.0

His 3.0

Lys 6.5

Arg 1.2

4. An isoform of soluble immune response suppressor protein according to claim 1 which is SIRS-α7 characterized by the following:

(b) focusing as a single entity at about pH 7.0 on preparative isoelectric focusing in a granulated gel; and

(c) a partial amino acid composition as contained in the following table:

Asp 8.45

Thr 4.66

Ser 5.51

Glx 13.00

Gly 11.97 Ala 11.93

Val 8.02

Met 1.87 lieu 5.69

Leu 9.20 Tyr 2.77

Phe 4.28

His 2.33

Lys 4.72

Arg 5.64

5. An isoform of soluble immune response suppressor protein according to claim 1 which is SIRS-α7 characterized by the following:

(b) focusing as a single entity at about pH 7.0 on preparative isoelectric focusing in a granulated gel;

(c) a partial amino acid composition as contained 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

He 5.0

Leu 8.1

Tyr 2.7

Phe 4.3

His 2.4

Lys 4.8

Arg 4.7 and

(d) having an amino-terminal sequence of

Met-Thr-Glu-Glu-Asp-Gln-Gln-Ser-Ser-Gln-Pro-Lys-

Thr-Thr-He-Asp-Asp-Ala-Gly-Asp-Ser-.

6. An isoform of soluble immune response suppressor protein according to claim 1 which is SIRS-87 characterized by . ^"

(a) a specific activity of >7.0 x 10 SU/μg of protein when assayed by in vitro antibody-forming cell response to SRBC;

(c) a partial amino acid composition as 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

He 3.3

Leu 5.9

Tyr 3.8

Phe 3.1

His 2.2

Lys 3.7

Arg 3.3

7. A pharmaceutical preparation suitable for parenteral administration for prophylaxis of organ rejection said preparation comprising an effective amount of at least one isoform of soluble immune response suppressor protein as claimed in claim 1 and a conventional pharmaceutical parenteral carrier material.

8. Antibodies raised against an isoform of soluble immune response suppressor protein as claimed in claim 1.

9. The antibodies of claim 8 which are monoclonal antibodies.