JPH0335782A - Soluble t11 protein which inhibits activation of t11-mediated t cell - Google Patents

Abstract

NEW MATERIAL: A soluble peptide having an amino acid sequence corresponding to a part of extracellular domain of human T11 glycoprotein capable of inhibiting T11-mediated T cell activation.

EXAMPLE: A soluble peptide having an amino acid sequence of the formula.

USE: Inhibition of T11-mediated T cell activation.

PREPARATION: The soluble T11 protein can be produced by fragmentation of enzyme, peptide synthesis or recombinant DNA technique.

COPYRIGHT: (C)1991,JPO

Description

[Detailed description of the invention]

The present invention relates to soluble peptides corresponding to portions of the extracellular domain of the human 1-Tll glycoprotein. The peptides can inhibit Tllll-mediated brain cell activation. T lymphocytes were originally identified by their ability to form spontaneous aggregates with sheep red blood cells (SRBC) [Brain et al. (1970) Clinical and Experimental Immunology (CIin). E
xp, Immunol, ) 6: 681-688;
Combs et al. (1970) International Archives of Allergy and Agelide Immunology (Int, Arch, Alle
rgyApp 1. Immuno +, ) 39: 6
58-663; Lay et al. (1971) Nature (London) 230=531-53
31゜The molecules that mediate this cell-cell interaction are 5
0 kDa T lymphocyte (T cell) specific Tl1 (CD2
) surface glycoproteins

(1981) Journal of Immunology (J, I)
mmunol, ) 6:2-117-2122: Kamoun et al. (1981) Journal of
Immunology (J, Immunol,) 127:987
-991: Verbi et al. (1982) European Journal of Immunology (Eur, J
, Immunol, ) ±2:81-86: Bernard (
Ber, nard) (1982) Journal of Experimental Medicine (J, Exp, Med,
) 155: l 317-1333;
(1984) Cell (Cel 1) 36:
897-906] and lymphocyte function-related antigen (LF
A-3) is a widely distributed membrane protein [Dustin et al. (1987) Journal of Experimental Medicine (J, Exp. Med. The homolog (TIITS) is expressed at high levels on 5RBCs [Munig et al.
986) European Journal of Immunology (Eur, J. Immunol,) 16:1615-
The adhesion function of 16211°Tll facilitates the necessary cell-cell interactions that occur during physiological T cell responses. By interfering with T cell adhesion (i.e., CD2-LFA-3 interaction), a monoclonal antibody directed against an epitope on Tll, called anti-Tll I [Meuer et al. (1984)] Cell (Cel +) 36: 897-9061 is
Inhibits the formation of T lymphocyte-5BRC rosettes, interferes with T lymphocyte activation, and disrupts thymocyte-thymine epithelial interactions [Shaw et al. Nature (0:/ton). )323:262
-254; Vollget et al. J. Immuno 1. 138: 358-363]. In contrast, anti-Tll! Two other anti-Tll antibodies, termed Tll and Tl1s, do not interfere with Tll-mediated adhesion but cooperate, leading to antigen-independent T lymphocyte activation [Meuer et al. (1984) Cells (Ce 11) 3
6: 897-906]. The primary structure of human Tll was explained by protein microsequencing and cDNA cloning.
Sayre et al. (1987) Proceedings of the National Academy of Sciences (Pr.
oc, Nat. 1, Aca, 5ci), USA 84:2941-2
945; Sews II et al. (1986)
Proceedings op National Academy
Op Sciences (Proc. Natol, Aca, 5ci), USA 83:87
18-8722; Seed et al. (1987)
Proceeding Obsession Adventure Academy
Op Sciences (Proc. Natol, Aca, 5ci), USA 84:33
65-33691, a protein deduced by cDNA sequence, is composed of three segments: (i) an extracellular segment, which accounts for more than half of the molecule; It has only limited homology to the membranes of immunoglobulin supergenes containing glycoproteins [Sayre et al.
987) Academy of Sciences (Proc, Nat)
o 1゜Aca, 5ci), USA 84:2941
-2945; Sewe 11 et al. (1986
) Proceedings of National Academy
・Op Sciences (Proc, Natol, A
ca, 5ci), USA 83:8718-8722
: Seed et al. (1987) Proceedings of the National Academy of Sciences, Natol, Aca.
. 5ci), USA 84:3365-3369]
(ii) a single characteristic hydrophobic transmembrane (
transmambrana) segment: and (
iii) 1 rich in proline and basic amino acid residues
17 amino cytoplasmic domain. Additionally, encodes the murine and rat equivalent of Tll (encod jn
g) The cDNA was cloned and sequenced to show >50% amino acid sequence identity [Clayton et al. (1987) European Journal of Immunology (Eur, J. Immunol). 1367-1370; Swe 11 et al. (1987) European Journal of Immunology 17: 1015-1020; Williams et al. (1987)
Journal of Experimental Medicine (
J, Exp, Med, ) 165: 368-380
: Clayton et al. (1988) Journal of Immunology (J, Immunol,) 1
40:3617 3621]. The present invention relates to soluble peptides corresponding to portions of the extracellular domain of the human Tll glycoprotein. The peptide may induce Tllll-mediated brain cell activation 11-m.
edited T-cell activation
ion) can be inhibited. The soluble peptides of the invention correspond to the amino terminal amino acid sequence of the extracellular domain of human Tit. In particular, the soluble peptide consists of about 100 amino acid residues, which correspond to the amino-terminal portion of the extracellular domain of human Tll, or a fragment thereof, which can impair Tllll-mediated brain cell activation. Soluble peptides of the invention can be made by enzymatic fragmentation, peptide synthesis, or recombinant DNA technology. They can be used to block T cell functions that depend on antigen activation. The present invention relates to a soluble peptide consisting of an amino acid sequence that corresponds to a portion of the extracellular domain of the human Tll glycoprotein and is capable of inhibiting Tllll-mediated brain cell activation. The peptides of the invention are soluble in aqueous media. In one embodiment, the soluble human 1-Tll peptide is a fragment of the extracellular domain of the human Tll glycoprotein. This fragment can be any part of the Tll extracellular domain that can inhibit T11-mediated T cell activation. In a preferred embodiment, the soluble peptide corresponds to the amino terminal portion of the extracellular domain of human Tll and is about 100 amino acids in length. Preferably, the peptide has the following amino acid sequence: NH. Lys Glu Ile Thr Asn Ala L
eu Glu Thr Trpll Gly Ala
Leu Gly Gin Asp Ile Asn L
eu Asp21 lie Pro Ser Phe
Gin Met Ser Asp Asp [1e31
Asp Asp IIs Lys Trp Glu
Lys Thr Ser A, 5p41 Lys Ly
s Lys IIs Ala Gln Phe Arg
Lys Glu51 Lys Glu Thr
Phe Lys Glu Lys Asp
Thr Dingyr61 Lys Leu Phe
Lys Asn Gly Tbr Leu Lys l
1e7] Lys His Leu Lys
Thr Asp Asp Gln Asp
l1e81 Tyr Lys Vat Ser口e
Tyr Asp Thr Lys Gly91 Lys
Asn Vat Leu Glu Lys Ile
Pha Asp LauOOH This soluble peptide with a length of about 100 amino acid residues is
It consists of the portion of the extracellular domain of Tll that encodes the epitopes of Tll+jt and Tllx, as well as a functional LFA-3 binding domain. The T l 1 l and T l l 2 epitopes are localized on soluble peptides. T111 and Tl
The l□ epitope is an antigenic determinant that is required to impair Tllll-mediated brain cell activation. The above amino acid sequence corresponds to the portion of the naturally occurring extracellular domain of Tll that can impair Tlll-mediated brain cell activation. The amino acid sequences included in the present invention are Tllll
Includes similar or homologous sequences that encode proteins that can mediate brain cell activation. Furthermore, the structure of the peptide may be affected by the deletion, addition, or addition of one or more acid residues in the sequence.
Peptides can be modified by inversion, insertion, or substitution to impair Tllll-mediated brain cell activation. All such modifications of the above amino acid sequences without reducing the properties of the peptide, ie, the ability of the peptide to impair Tllll-mediated brain cell activation, are encompassed by the present invention. Naturally occurring allelic changes and modifications are encompassed within the scope of the invention, so long as the changes do not substantially reduce the ability of the peptide to impair Tllll-mediated brain cell activation. The present invention also provides a DNA sequence encoding a soluble human Tll peptide that corresponds to a portion of the extracellular domain of human Tll that can inhibit Tllll-mediated brain cell activation, particularly the 100 amino acid sequence shown above or its substantial decytosis. Concerning equivalents. The DNA sequence contains nucleotide deletions,
Modified by insertions or substitutions, it exhibits substantially the same properties as the above peptide of about 100 amino acid residues. All such modifications of the DNA sequence are within the scope of the invention, so long as the DNA sequence encodes a soluble peptide capable of inhibiting T11-mediated T cell activation. DNA of the present invention
Sequences can be synthesized chemically or by recombinant DNA technology. The present invention further relates to an expression vector consisting of a DNA sequence encoding a soluble human Tll peptide that corresponds to a portion of the extracellular domain of human Tll that is capable of inhibiting Tllll-mediated brain cell activation. Preferably, the expression vector consists of a DNA sequence encoding a peptide of about 100 amino acid residues corresponding to the amino-terminal portion of the extracellular Tll domain. Amino acid residues can be deleted without essentially reducing the properties of the peptide.
Modifications of the peptide, such as insertions or substitutions, are encompassed by the invention. The invention further relates to cells transformed with the above expression vectors. Soluble peptides of the invention can be made by tryptic fragmentation of the human Tll glycoprotein or portions thereof, by peptide synthesis or by recombinant DNA techniques. Preferably, the soluble T11 peptide is Tllll
It will be generated by inserting into an expression vector a DNA encoding a peptide sequence capable of mediating brain cell activation (eg, Tll DNA representing the desired amino-terminal portion of the extracellular domain of Tll). The transformed cells then express a soluble human Tll peptide that corresponds to the portion of the extracellular domain that can impair Tllll-mediated brain cell activation. In addition to genetic engineering techniques to synthesize the soluble peptides of the present invention, soluble peptides can be directly synthesized by chemical protein synthesis procedures. For example, about 100 amino acid sequences or modified equivalents thereof may be
Synthetic leather can be made by the solid phase procedure of ld). The present invention further provides a method for inhibiting T11-mediated T cell activation, comprising administering to a patient a soluble peptide consisting of an amino acid sequence corresponding to a portion of the extracellular domain of the human Tll glycoprotein that is capable of inhibiting T11-mediated brain cell activation. Concerning methods of inhibiting. Preferably, the soluble peptide consists of a sequence of about 100 amino acid residues corresponding to the amino-terminal portion of the extracellular domain of the human Tll glycoprotein, or a fragment thereof, which is capable of inhibiting Tllll-mediated brain cell activation. In one embodiment of this method, the soluble peptide can be administered intravenously. Soluble Tll peptides of the invention will generally bind to human lymphocytes and human red blood cells that express a homologous set of surface structures. Soluble peptides can also be
the ability of a lymphocyte to compete with Tll naturally present on the surface of human lymphocytes and thus contact its target cells (when the lymphocytes are cytolytic cells);
Alternatively, it interferes with the ability of lymphocytes to contact macrophages with Tll-binding structures that allow cell-to-cell contacts necessary for proliferation. To test soluble peptides for their ability to inhibit lymphocyte proliferation or cytotoxic effector function, soluble peptides are contacted with lymphocytes prior to stimulation with mitogens, and the extent of proliferation is determined using standard techniques. , measure, and compare the results to a control without soluble peptide. The soluble peptides of the invention can be used in a variety of diagnostic and therapeutic applications where the T11 surface glycoprotein is expressed on the surface of many human T cell malignancies, such as T cell leukemia and lymphoma. Additionally, autoimmune diseases such as rheumatoid arthritis and systemic
Lupus eltomatosis (Systemic Lup)
USErthomatosis (SLE) is characterized by the presence of large numbers of Tll-supporting T cells in the blood and lymph. Rapid cell turnover in these disease states can shed Tl1 molecules into the blood stream. The Tll soluble peptides of the invention can be used as immunogens to generate polyclonal or monoclonal anti-Tll antibodies according to conventional techniques. These antibodies can be labeled with any conventional label, such as a radioisotope, and used in conventional immunoassay methods to measure serum Tll levels and thus monitor patients with T cell-related diseases. can. A particularly sensitive ELISA-type assay would use two anti-Tll antibodies, each directed against a different epitope on the surface of Tll, in a sandwich format. Disease states for which the soluble peptides of the invention can be used include excess T, which plays an important role in amplifying the immune response.
Includes medical conditions characterized by undesirable activity of the immune system resulting in activation of cells. These conditions include: SLE, early-onset diabetes;
Multiple sclerosis; allergic conditions: inflammatory conditions such as edema, ulcerative colitis, inflammatory bowel disease, and Cohn's disease; and allograft rejection (eg, rejection of a transplanted heart or kidney). Soluble Tll peptides compete with surface bound Tll and its ligands on target cells, thus reducing the amplification of the immune response. Soluble Tll peptide mixed with a pharmaceutically acceptable carrier, e.g., physiological saline, is administered intravenously to a human patient in an effective amount, e.g., 20 μg to 500 μg/kg body weight. Preferably, the soluble peptide mixed with a pharmaceutically acceptable carrier consists of about 100 amino acid residues corresponding to the amino terminal portion of the extracellular domain of Tll. For some conditions, soluble Tll peptides can be administered directly to the site of greatest need; for example, soluble Tll peptides can be injected directly into the inflamed joints of human patients suffering from rheumatoid arthritis. The invention will be further illustrated by the following examples. Examples Terms used in the examples T11. is defined herein to be a soluble protein having an amino acid sequence encoding the entire extracellular domain of the Tll glycoprotein. 'rtis protein is also known as Tll
Transmembrane that does not interfere with protein solubilization (
tyansumambrane). See, Reinherz, US Patent Application No. 932.871, filed November 18, 1986, the teachings of which are incorporated herein by reference. The Tll cDNA PH1 [which was the source of this DNA] was digested with PstI and PvuII, blunt-ended with T4 DNA polymerase, and then the cytidine-containing oligonucleotide, CTAAGGAT
Combined with CCTTAGl, the last nucleotide for serine at position 178, and GGATCC (B
amHI recognition sequence) was reconstructed. The plasmid preparation was then digested with BamHI and the truncated Tll
cDNA fragments were separated by gel purification. Then c
pAc 373 vector in which the DNA fragment was digested with BamHI, BamHIL, and Escherichta
was used to transform E. coli DH5 cells. Recombinant clones were isolated and their construction confirmed by restriction endonuclease mapping and M13 sequencing. Oligonucleotide synthetic skins used standard cyanoethylphosphoramidite chemistry. T11. Array of Autographers California
phacalifornica) nuclear polyhedrosis (
polyhedorosis virus (AcNPV)
The metastasis in the middle was as described [Smith et al. (1985)
Op National Academy Op◆Sciences (
Proc, Natol. Aca, 5ci), USA 82:8404-840
8: Husey et al. Nature
e) (London) 331:78-81]. Tl 1. of

[3.S] Methionine labeling and large-scale purification
It was as described [Hussey
ey) Nature (London) 33
1:78-81] but Ann Egel (Affi-
Anti-T11. (3T4-885)
It was used. Fermentation of trypsin 70 mmol glycine/240 mmol Tris-H
Purified T11, (200 μg) in C1 (pH 8,0)
/ml) was added to a final concentration of 6 mmol dithiothreitol in a stoppered tube for 30 minutes at room temperature and then diluted with 21 mmol iodoacetamide in the dark.
Amidomethylated for 0 minutes. The sample was then made up to 4 mmol in CaCl on ice and L-1-tosylamide-2-phenylethyl chloromethyl ketone-treated trypsin was added to bring the protein/enzyme ratio to 50.
0:1 (wt/wt). Samples were removed at 0 minutes and mixed with N'aDodSO, sample buffer, and the remaining digestion mixture was removed at 10 and 35 minutes and mixed with NaDodSO, sample buffer and NaDodSOa/
Mixed for PAGE. Preparative high performance liquid chromatograph 4-(HPLC) HP
Purified 'rl l5 (110 μg) for LC analysis.
was subjected to gentle reduction and amidomethylation, followed by microcentrifugation for 10 minutes. The sample (300/J (1)
mm). The material was purified at 0.5 ml/min in 0.2 molar sodium phosphate buffer (pH 6.5) using a Hewlett-Packard 1090 liquid chromatograph equipped with a diode array detection monitor. It was eluted with minute 11 (0,5
m1) and subsequently non-reducing NaDodSO,/
Analyzed by PAGE. The reduced and alkylated T l l s were dialyzed into isotonic phosphate buffered saline (PBS). This material (500μgs2.14I1g/m
I) in 37 cases, L-1-) sylamide-
Digested with 2-phenylethylchloromethylketone-treated trypsin (lpg) for 60 minutes. Digestion was stopped by the addition of soybean trypsin inhibitor (lpg), and the microcentrifuged samples were incubated with H
It was applied to PLC. Molecular weight markers were subjected to silica gel chromatography under the same conditions [monoclonal mouse IgG (150 kDa), bovine serum albumin (
69kDa), ovalbumin (46kDa), carbonic anhydrase (30kDa), and cytochrome C (12,4kDa)]. Peak I derived from tryptic digestion T l s in 0.2 molar sodium phosphate buffer (pH 6°5)
The purified material (30 μg) from I (see Figure 3C) was adjusted to pH 8, by addition of a predetermined amount of 1 molar.
Adjusted to 2. The sample (440 μa) was then heated to 1 mC
1 freshly dried 1! J-marked Polton-Hunter (B
Olton-Hunter reagent [New England
d Nuclear), 4400Ci/mmol;
4 in a vial containing 37 GBq)
℃ for 15 minutes. 1 mol of Tris-MCI (
1) An aliquot (15 μl) of H7,6) was added to the mixture, the mixture was then dialyzed at 4°C in 2 kDa cutoff dialysis tubing, and further gelatin (
Desalted with 5 ml of Bio-Gel P-2 (200-400 mesh) equilibrated with PBS containing 1 mg/ml) and 0.05% sodium 7) diide. The radiolabeled fragments were transferred to Protein A-Sepharose (S
monoclonal antibody (3) bound to epharose)
T48B5 and 1old24c1) were immunoprecipitated for 16 hours at 4°C. The beads were washed 4 times with 10 mmol of Tris-HCl (pH 6,8) and the radiolabeled fragments were washed with 10 mmol of glycine hydrochloride (pH 2,0).
) and immediately add 0.2 volumes of 1M Tris-HC.
l (pH 7,6) and then mixed with an irrelevant carrier peptide (l mg/ml). Routinely 20-25% of the total radiolabeled fragments were immunoprecipitated. Purified radiolabeled fragments were immunoprecipitated. The purified radiolabeled fragment was treated with endoglycosidase F [New England Nuclear
d Nuclear)], Luesh+-(Luesh
er) and Bron% Journal of Immunology (J, Immunol), 1
34:1085-1089 (1985), and was run on a 15% polyacrylamide gel in NaDodSOa/PAGE. Rosette-inhibition assay (Jurkat) cells were washed in minimal essential medium for suspension culture and 2% (vol/v. l) fetal calf serum history washing medium) and incubated. Resuspend at 10'/m+. Bank 5RBC (h
a n k) balancing; washed twice with physiological saline and resuspended at 5% (vol/vol) in washing medium; 10μa 5RBC times 12 75m
50 μA of washing medium, ovalpmin (72-312 °C g/ml), or l 5 m plastic tubes.
Tll5 fragment for kDa (72-312°C g/ml)
was added and incubated for 30 minutes at 4°C. Subsequently, 20 μQ of Jharkad cells were added, incubated for 5 minutes, centrifuged in a Sorva II RT6000 at 800 rpm, and incubated for 1 hour at 4°C. Cell mixtures were gently resuspended, mounted on glass slides with coverslips, and rosette formation was assayed with a Zeis S light microscope. 5RBCs were 3-5 μm in diameter. Generation and Characterization of Soluble Recombinant Tl1 Molecules To generate the secreted form of Tl1 molecules, PB2 cD
NA [Seire et al. (1987) Proceedings of the National Academy of Sciences (Proc. Natol, Aca, 5cl), USA 84:29
41-2945] was truncated as described in Figure 2 and attached to a synthetic linker so that the predicted 185 amino acids of the mature extracellular segment were preserved (Figures 1A and 2A). After cloning into the pAc373 transfer vector, the truncated Tll cDNA construct was integrated into the AcNPV genome by homologous recombination [
Smith et al. (1985) Proceedings of the National Academy of Sciences (Proc, Natol+Aca, 5ci)+USA
82:8404-8408; 7 Husse
y) et al., Nature (London) 33
1:78-811° pure recombinant baculovirus, termed TI Is AcNPV, was used to infect Spodoptera frugiperda (Spodoptera frugiperda).
frugiperda (SF9) cells were infected. SF9 cells were infected with Tlis AcNPV or Taha wild-type AcNPV and then assayed (Hussey
) et al., Nature 331:
78-81 (1988) in sIS methionine. The products of anti-Tll, (3748B5) immunoprecipitation were examined by autoradiography on NaDod-SOa/12.5% acrylamide mini-slab gels. In FIG. 2B, lanes a and b show material immunoprecipitated under reducing conditions from the supernatant of TI Is AcNPV-infected and AcNPV-infected SF9 cells, respectively. Lanes C and d are T 11 s AcNPs, respectively.
Material immunoprecipitated under non-reducing conditions from lysates of V-infected and AcNPV-infected SF9 cells is shown. Lanes e and f are Tl1s AcN, respectively.
Material immunoprecipitated under non-reducing conditions from supernatants of PV-infected and AcNPV-infected SF9 cells is shown. Analysis of material produced by metabolic radiolabeling of Tl1s AcNPV-infected SF9 cells was performed at 48 p.i.
- The major secreted product at 72 hours was T l
l s (data not shown). The major -Is-labeled secreted material seen by reducing NaDodS O4/PAGE migrates as poorly separated dimers and heptanomers, as seen in metabolic radiolabeling experiments ( *Figure 2B, lane e). Under reducing conditions, Ti1l is a doublet of 28 kDa, accompanied by a trace of a doublet of 25 kDa, and an additional protein band is present due to the Coomassie blue coloration. I didn't. Reduced and purified 28kDa doublet (200 pmol)
, polyvinylidene difluoride membrane [Millipore (Mi
111pore); pore size, 0.45 μml;
Matsudaira et al., Journal of Biological Chemistry (J, Bio 1. Chem,) 262:
0035-10038 (1987) and by the method of Coomatsey (Co.
omassie) blue colored substances were measured using an Applied Biosystems (Applied Biosystems) equipped with an online 120A biotechnylthrohydantoin analyzer.
Bi. The amino-terminal sequence of T l l s was confirmed by amino-terminal sequencing using the 03RPTH program on a Peptide Sequencing System Model 470A. The first nine cycles are Lys-Glu-11e
produced a single distinct sequence -Thr-Asn-Ala-Leu-Glu-Ehr and established that this substance is the product of the Tll gene [Sayre et al.
987) Proceedings of the National Academy of Sciences (Proc, Nat. 1, Aca, 5ci) - USA 84:2941-2
945; Sewe 11 et al. (1986) Proc-Natol, Aca, 5
ci)-USA 84:8718-8722; Seed et al. (1987)
roc. Natol, Aca, 5ci), USA 84:33
6533691 And furthermore, NaDodSo4/P
We demonstrated that each of the components of the doublet seen at 28 kDa by AGE consists of the same polypeptide backbone and differs from each other, probably as a result of post-translational modifications. Interestingly, when the substance migrating at 25 kDa was also amino-terminally sequenced, it showed the same amino-terminus as above. Thus, the 25 kDa material represents an amino-terminal fragment of T l l fi that has lost some of its C0OH terminal residues. Moreover, the results show that the baculovirus expression system has the ability to correctly cleave the signal peptide from the T l l m precursor. Alligator, 1a (10"JI cells) of SF9 cells produce 1 mg of secreted Tl 18 over a 72 hour culture period. Figure 2C shows analysis of large scale preparation and purification of Tl1s. Aliquots of purified T l 1 m were prepared unreduced (lane a), mildly reduced and alkylated (lane b), or fully reduced (lane C).
The morphology was examined in mini-slab gels by staining the material with Coomassie blue. The molecular weight markers used in B and C were phosphorylase b (97,4 kDa), bovine serum albumin (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa), and lactoglobulin A.
C18,4kDa). As shown in Figure 2C, lane b1, mild reduction and subsequent alkylation are sufficient to polymerize dimeric TIIm, where such a polymer is only 28 kDa under non-reducing conditions. The original and alkylated Tl1s were purified using monoclonal antibody 3PT2H9 (anti-Ti
1l), 10Id24C] (anti-Til or Imo
no2A6 (anti-Ti1l) (data not shown), anti-T11+ and anti-T11. immunoprecipitated both forms of Tl1s, but
Anti-Tl1s did not immunoprecipitate either form, indicating that, independent of reduction and alkylation, Tl1m does not immunoprecipitate additional T111 epitopes (3PT2H
9) and the Tl1n epitope, but the T11* epitope was not present either before or after reduction and alkylation of T115. The absence of the TI Is epitope was not a result of the baculovirus system's inability to produce and process human proteins. This is because expression of full-length transmembrane Tll in SF9 cells infected by baculovirus carrying the PBI cDNA has been shown to yield a protein that reacts with antibodies to T113. [AIcover et al., European Journal of Immunology (Eur. J. Immunol.), Vol. 8: 363-367 (
1988)]. Analysis of Fragmentation of Tlls To more precisely localize functional Tll epitopes within the 7113 polypeptide, purified Tlls
was partially digested with trypsin. Figure 3A shows tryptic digestion of Tl1s. The sample is reduced to reducing Na D o d SO4/PA
Analyzed by GE in a 15% acrylamide Minnie Game Lap gel. Lanes aS b and C are 0, 10 and 35 min digests, respectively. Colored bands >60 kDa are derived from 2-mercaptoethanol I; non-protein artifacts. This TI Is preparation is a naturally cleaved fragment, with Na
DodSO, 25kDa on polyacrylamide gel
(Fig. 3A, lane a). Examination of the samples by NaDodSO4/PAGE at various times of tryptic digestion shows that after short digestion (5 min) the amount of 25 kDa material increases with respect to 2 g kDa material, and a trace amount of 15 kDa is evident. (data not shown). After intermediate digestion (10 min), only a small amount of 28 kDa material survived and equal amounts of material at 25 kDa and 15 kDa were present (Figure 3A, lane b). After extended digestion (35 min), the majority of material is present at 15 kDa (Figure 3A, lane c)-2
The 5 kDa and 15 kDa fragments were obtained from Coomatsie (C
oomassie) was divided as a double line on the blue colored gel. Electrical plotting and lead <2
Microsiefencing of the 5 kDa and 15 kDa fragments revealed distinct sequences for each, Lys-G+u
-I 1e-Thr-Asn-Ala-Leu-G 1
Generate u-Th r. Thus, both fragments are derived from the amino-terminal region of the 711m molecule.
The kDa fragment cleaves more extensively at the COOH terminus than the 25 kDa. HPLC analysis of TtIs and derived fragments Gently reduced and amidomethylated TtIs in a non-dissociating buffer at neutral pH was detected as a trailing edge as a 60 kDa species from molecular sieve HPLC.
A peak with a ling edge) was eluted, but a peak with a
Figure 3B, peak III), the preparation is NaDodSO4
28 kDa species and trace amounts of 25 kDa by /PAGE
It was dug from the seed of a. NaDodSO4/PAGE (data not shown) of the fractions at the 60 kDa HPLC peak showed that this material migrated as a 28 kDa species and that the material at the trailing edge was 25
It was shown to contain both a kDa species and a 28 kDa species. Peak I (excluded material) and Peak I
Similar analysis of material from I showed that both peaks contained the 28 kDa species in oligomeric form. The material from peaks IV and V was not a protein and was probably a low molecular weight UV absorbing material. Thus, -wood-like TI Is are either non-covalent homosymers or have similar molecular masses.
It is an elongated molecule with a hydrodynamic radius that is larger than that of a spherical molecule with a spherical molecule (ar mass). The putative noncovalent T l l * is a mild reduction followed by carboxymethylation (which can destabilize noncovalent interactions with charged carboxyl groups)
and 0.2 mol glycine hydrochloride (pH 3,0
) was not depolymerized by molecular sieving. Thus, non-covalent interactions between Tlls, if present, are not easily destabilized and directly after affinity chromatography, purified T11. The reducing and alkylating properties are not destabilized, as shown by a similar molecular sieve test. Additionally, reduced and alkylated T1 on a column of HPLC molecular sieves! The behavior of tryptic fragments derived from , is also surprising. Extended tryptic digest non-reducing NaDodSO
a/PAGE shows one major 15 kDa component and a small 25 kDa component (equivalent to that seen in Figure 3A, lane C), but the elution profile of the sieve column (Figure 3C) shows a major ~35 kDa component. and about 14
kDa peaks (peaks II and III, respectively)
and a small 40 kDa peak (Peak I). The non-reducing NaDo d of the material from these peaks
SO4/PAGE is 40kDa (peak and 3
The 5 kDa (peak II) material has an apparent molecular mass of 25 and 15 kDa, respectively, and the other material eluting after peak III is completely absorbed into the vegutide, which is too small to appear on the colored slab gel. Indicates that it has been destroyed. As concluded from these data, the chromatographic behavior seen using Tl1s is similar to its 25kD
a* and a 15 kDa fragment, suggesting that the structure of recombinant T l 1 * and perhaps native Tl1 molecules is elongated or predisposed towards non-covalent oligomerization. Whether this behavior has any relevance for the physiological function of native T11 remains to be established, but it appears to have. Analysis of the Tll epitope Reducing NaDodSO4/PAGE of the 28 kDa T11s and tryptic fragments and subsequent immunoblotting did not destroy the Ti1l epitope recognized by monoclonal antibody 3PT2H9 (data not shown). (not shown) actually abolished the ability of anti-monoclonal antibody 3T48B5 to bind, explaining that these T11 epitopes are not identical. To reveal which Tll epitopes are present on the 15 kDa amino-terminal tryptic fragment, the peak! The material (Figure 3C) was purified on a column of HPLC molecular sieves, and the material was radiolabeled with Polton-Hunter reagent. The ability of individual monoclonal antibodies to immunoprecipitate Jn■-labeled Tl1fi was determined by Na
Assayed by D o d S 04/PAGE. Figure 4A shows radiolabeling and deglycosylation of the 15 kDa tryptic T113 fragment. Lanes a and b show glyphsylated and deglycosylated fragments, respectively. Lanes c, e and g are anti-T11. (3748B5), anti-T
11! (1o1d24CI) and Tl 1x(Irn
Figure 3 shows the glycosylated fragment recognized by the monoclonal antibody of (ono2A8). Lanes d, f and h show Tlts fragments in the supernatant remaining after treatment of the glycosylated fragments with the monoclonal antibodies described for lanes c, e and g, respectively. Molecular mass markers and NaDodSOa/PAGE are
It is like that in @, and this includes cytochrome c
(12,4 kDa) and aprotinin (6 kDa) were added. Figure 4A shows anti-Tl 1. (3748B5)
(lane C) and anti-T 112 (lane e) at 15
The kDa fragment is selectively immunoprecipitated, but the anti-T l 1
3 (lane g) indicates not allowed. Treatment with endoglycosidase F reduces the apparent molecular mass of the radiolabeled 15 kDa material to approximately 12.5 kDa (Figure 4A, lane b), which is due to the removal of one carbohydrate moiety from the 15 kDa fragment. Match. This treatment involves 3T48B5 (anti-T), which reacts with the polypeptide.
11l) or Iold24C1 (does not affect the ability of anti-Tll antibodies (data not shown), indicating that carbohydrates are not required for recognition by these antibodies. kDa Tl1s) expressed on a lipsin fragment, and anti-Tl1
Once the antibody was shown to inhibit the formation of 5RBC rosettes with human T lymphocytes, HPLC-purified T
It was important to determine whether the 15 kDa fragment of 1 s. 1 of Tl1i in the amount of microdulum
Upon pre-incubation with the 5kDa fragment,
Rosette formation is completely inhibited (Figure 4C). A similar amount of control protein (opalbumin) was found in 5RBC
failed to inhibit rosette formation (data not shown). Equivalent Embodiments Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many embodiments that are equivalent to the specific embodiments of the invention described herein. Such equivalent embodiments are intended to be encompassed within the scope of the claims. The main features and aspects of the invention are as follows. A soluble peptide consisting of an amino acid sequence corresponding to a portion of the extracellular domain of human Tll glycoprotein that can inhibit 1% T11-mediated T cell activation. 2. The soluble peptide according to item 1 above, consisting of about 100 amino acid residues corresponding to the amino-terminal portion of the extracellular domain of human Tll glycoprotein or a fragment thereof, capable of inhibiting Tll-mediated T cell activation. 3. Amino acid sequence: Lys Glu Tie Thr Asn Ala L
eu Glu Thr Trpll Gly Ala
Leu Gly Gin Asp Ile Asn L
eu Asp21 IIs Pro Ser Phe
Gin Met Sar Asp Asp 11e31
Asp Asp rle Lys Trp
Glu Lys Thr Ser Asp
41 Lys Lys Lys Ile Ala
Gin Phe Arg Lys Glu51 Ly
s Glu Thr Phe Lys C1u Lys
Asp Thr Tyr61 Lys Leu P
He Lys Asn Gly Thr Leu Ly
s 1ie71 Lys His Leu Lys
Thr Asp Asp Gin Asp 1ie81
Tyr Lys Val Ser Il,e
Tyr Asp Thr Lys Gly9, I
Lys Asn Val Leu Glu Lys
2. The soluble peptide according to item 2 above, which comprises Phe Asp Leu and a modified version of the peptide in which amino acid residues are deleted, inserted or substituted without essentially reducing the properties. 4. The soluble peptide according to item 3 above, wherein the fragment is purified by tryptic digestion of the extracellular domain of human) Tll glycoprotein. 5. T 11 r and T localized on the peptide
L xt shrimp! The soluble peptide according to item 3 above, consisting of -. 6. The soluble peptide according to item 5 above, which has an LFA-3 binding domain. 7. Human T11$ can inhibit Tll-mediated T cell activation
Part of the extracellular domain of the protein: DNA sequence encoding the corresponding soluble peptide. 8. A DNA sequence encoding the amino acid sequence set forth in item 3 above or a substantial decoding equivalent thereof. 9. An expression vector consisting of a DNA sequence encoding a soluble peptide corresponding to a portion of the extracellular domain of human T11 glycoprotein capable of inhibiting Tll-mediated T cell activation.
(1111111e Thr Asn Ala Leu
Glu Thr Trpll 'Gly Ala L
eu Gly Gln Asp lle Asn Le
u Asp2111e Pro Ser Phe Gl
n Met Ser Asp Asp 1ie31 A
sp Asp Ite Lys Trp Glu Ly
s Thr Ser Asp41 Lys Lys L
ys lie Ala Gin Phe Arg Ly
s Glu51 Lys Glu Thr Phe L
ys Glu Lys Asp Thr Tyr61
Lys Leu Phe Lys Asn Gly T
br Leu 1. ys l1e71 Lys His
Leu Lys Thr Asp Asp Gln
Asp Ice81 Tyr Lys Val Sar
Ile Tyr Asp Thr Lys Gly9
1 Lys Asn Vat Leu Glu Lys
Expression vector 11 encoding a soluble peptide consisting of Pha Asp Leu and a modified version of the peptide in which amino acid residues have been deleted, inserted or substituted without essentially reducing the properties, as described in item 1O above. Cells transformed with the expression vectors described. 12. Human Tll that can inhibit Tll-mediated T cell activation
A method for inhibiting T cell activation, comprising the step of administering to a patient a soluble peptide consisting of an amino acid sequence corresponding to a portion of the extracellular domain of a protein. 13. The soluble peptide has the amino acid sequence: Lys
Glu lee Thr Asn Ala Leu G
lu Thr Trp] I Gly Ala Leu
Gly Gln Asp Ile Asn Lau A
sp2111e Pro Ser Phe Gin M
et Ser Asp Asp l1e31 Asp
Asp Ile Lys Trp Glu Lys T
hr Ser Asp41 Lys Lys Lys
lie Ala Gin Phe Arg Lys G
lu51 Lys Glu Thr Phe Lys
Glu Lys Asp Thr Tyr61 Lys
Leu Pha Lys Asn Gly Thr
Leu Lys ll571 Lys His Leu
LY9 Thr Asp Asp Gln Asp
1ie81 Tyr Lys Val Ser lie
Tyr Asp Thr Lys Gly91 L
ys Asn Vat Leu Glu L
ys lie Phe Asp Leu and,
13. The method according to item 12, comprising a modified version of the peptide in which amino acid residues are deleted, inserted or substituted without essentially reducing the properties. 1'4. The method according to item 13 above, wherein the soluble peptide is administered intravenously.

[Brief explanation of drawings]

FIG. 1 shows the amino acid sequence and exon structure of human Tll protein. The amino acid sequence of human Tll, deduced from cDNA and genomic clones and its relationship to the five exons of the 12 kilobase gene on the chromosome, is given in (B). The 178 residues of the mature, secreted, soluble recombinant Tl1 molecule produced in the baculovirus system are shown in italic type. Figure 2 shows anchor-minus (anchor-minu 5) Tll in baculovirus vectors.
The organization of DNA and the production of secreted Tll are shown. Figure 2A shows the construction protocol for generating and cloning truncated Tll cDNA into the pAc373 transfer vector. FIG. 2B shows T l l
sノ [”S] Indicates the marking of methi f=n. 2nd C
The figure shows the large scale preparation and purification analysis of T l s. Figure 3A shows tryptic fragmentation of Tl1s. Figure 3B shows gently reduced and alkylated T
Figure 3 shows an HPLC chromatograph of l 1 g peptide. The third CrM is trypsin fragmented and reduced;
and HPLC chromatography of alkylated Tll. Figure 4A shows radiolabeling and deglycosylation of the 15 kDa tryptic Tlls fragment. Figure 4B shows the formation of rosettes between cells and 5RBCs for Jurkat T cells. Figure 4C shows the T of purified 15 kDa trypsin.
Figure 3 shows inhibition of rosette formation by the l l s fragment. t=lam)(L

Claims (1)

[Scope of Claims] 1. A soluble peptide consisting of an amino acid sequence corresponding to a portion of the extracellular domain of human Tll glycoprotein that is capable of inhibiting Tll-mediated T cell activation. 2. A DNA sequence encoding a soluble peptide corresponding to a portion of the extracellular domain of the human Tll glycoprotein that can inhibit Tll-mediated T cell activation. 3. An expression vector consisting of a DNA sequence encoding a soluble peptide corresponding to a portion of the extracellular domain of the human Tll glycoprotein capable of inhibiting Tll-mediated T cell activation. 4. A method for inhibiting T cell activation, which comprises administering to a patient a soluble peptide consisting of an amino acid sequence corresponding to a portion of the extracellular domain of human Tll glycoprotein that is capable of inhibiting Tll-mediated T cell activation.