JPH01501939A - Immunosuppressive peptides and usage - 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] Anti-epidemic peptides and usage Background of the invention Immunosuppression, non-regenerative anemia and neutropenia caused by persistent retrovirus infection It is the main clinical symptom in animals. Feline leukemia virus (FLV) also persists. can induce aplastic anemia and immunosuppression in viremic cats. It is a leukemia-inducing retrovirus.

Human acquired immune deficiency syndrome (AIDS) is caused by human immune deficiency viruses such as H IV-18 and HIV-17, hereinafter collectively referred to as HIV, caused by a virus. HIV also occurs in viremic individuals. and cause immunosuppression. Retroviruses in feline leukemia and human AIDS S-mediated immunosuppression in fact makes one susceptible to opportunistic infections. immunosuppressed It is these infections that most frequently cause morbidity and mortality in individuals. be. In FLY-induced disease, immunosuppression is at least partially due to p15E, That is, it is caused by viral gene products called transmembrane proteins. It seems like that. In AIDS, immunosuppression suppresses HIV replication in helper T cells. may be the result of a complex sequence of events related to production. However, )HI The presence of immunosuppressive peptides in V (described below) may inhibit gp41, p15E HIV immunity. Anti-inflammatory analogues may also be used in immunosuppression associated with early establishment of HIV infection. suggests playing a certain role.

feline leukemia virus The complete viral protein; can exert in vitro immunosuppressive effects similar to those induced by viruses. [C1ancjolo et al., Science ce), 230=453.1985], one measure of this immunosuppressive effect is In vitro inhibition of gen-induced lymphocyte proliferation. C1anciolo The peptide disclosed by ) et al. is derived from MLV and has the amino acid sequence LQNR RGLDL IFLKEGGL 0 This sequence with some other retrows When compared to the sequence of the transmembrane white matter of the virus, this region shows that FLY, homologous T cells Lymphotrophic viruses (HTLV-1 and II), bovine leukemia virus (BL V), and to a very small extent between retroviruses, including HIV. Indicates that it is well preserved. Cianciolo et al. The murine p15E peptide is a carposi cross-linked to bovine serum albumin (BSA). reported that it is an active immunosuppressant only when it is an imide.

Antibiotics in various cell lines derived from human cancers by fluorescence-activated cell sorting A monoclonal against an uncharacterized antigenic determinant of p15E that detects Antibodies may also be used as described [in the Journal of Experiments - J. (J, Exp, Med, 159:964.1984), L Kashinaga et al. did not disclose the biochemical properties of p15E-related antigens, and that, 3 5.000 Dalton protein or by various human leukemia and cancer cell lines. No relationship has been shown between the expressed f:110.000 dalton proteins; And it's not listed there. In vitro inhibitory activity using monoclonal antibodies These antibodies are highly sensitive to viral p15E. binding to conserved immunosuppressive regions or that such binding causes immunosuppression. Blocking is not shown. In fact, the authors spoke orally at a recent meeting. The antibody reportedly contains a 17-amyl acid peptide from a conserved region of p15H. Does not bind to Tido. Recent reports also indicate that biologically active p15E-related antigens are released by cell lines derived from human tumors [Journal of Immunology] (J, Im1m+uno1.), 137:2726.1986].

In some ways, human leukemia is caused by a synthetic immune system similar to the immunosuppression that retroviruses induce in China. Often characterized by hemosuppression. Cell lines derived from human leukemia, all In other words, 562 and HL-60 cells are grown by in vitro proliferation of normal bone marrow cells and Reported to secrete factors that block mitogen-induced differentiation of tile cells . Additionally, some of these cells, namely K563 and HL-60, were It has been reported that in vitro treatment with a cell differentiation agent reduces the expression of repressor factors. Follow up The following documents can be cited as additional information: Olfsson, T, Olsson, ■, ( 1980), a leukemia-associated inhibitor derived from a human promyeloid cell line (HL-60). In vitro suppression of normal granulocyte formation by (LIA), leukemia research (Lauke mia Res, ), 4:437゜Olfsson, T, , Elsin (Nilsson), E, , Ortho”/ (Olsson),! (1984) in myeloid leukemia producing leukemia-associated inhibitor (LAI). Cell characterization and demonstration of LAI-producing cells in normal bone marrow, leukemia research ( Leukemia Res, ), 8:387゜ S, Tetnberg, H-N-, Chiadosogero (7 gigs.

glou) % A, S -, Robinson, S, H , (1985), by leukemic cell lines after chemically induced differentiation. Loss of suppression of normal bone marrow colony formation. Blood, 65: 1 00゜Chiao, G, W, Heil, zo, Le Lutton, J.D., Choi N.Y.S., Leu. Leung, K. (1986), Inhibition of leukocytes by leukemia induction inhibitors. Activation and inhibition of function, PNAS, 83:3432° The purpose of the present invention is to inhibit the immunosuppressive peptide region of p15H and p15E itself. The objective is to provide corresponding antibodies.

Another objective is to develop peptides that can be used as vaccines against FLY. That is.

Still other objects are immunosuppressive peptides and the like for therapeutic applications. The purpose of the present invention is to provide antibodies that

The objective of the present invention is to prevent viral, non-viral, or endogenous viral infection. antibodies for detecting p15E-related immunosuppressive factors induced by viruses. It is to provide.

Summary of the invention When conjugated to carrier molecules, immune-related disorders such as autoimmune diseases, transplant rejection and Novel immunosuppressive peptides that can be used as therapeutic agents in treating allergies An array is provided. Also, retroviruses, such as FLV%BLV, HTLV , and) (vaccines to prevent immunosuppression and disease when exposed to IV It is provided that these peptides are used as peptides. In addition, immunoassay Diagnosis of human cancer by identifying the expression of p15E-related proteins in Antibodies against the inhibitory peptide are provided.

The present invention also provides biologically active, soluble immunosuppressants without cross-linking to carrier molecules. Provide peptides. contained within p15E (designated 16B) and the amino acid One peptide with the sequence AKLRERLKQRQQ exhibits unusual adhesive properties . The 16B peptide antiserum was found to neutralize the infectivity of the virus.

Blocking antiserum with 16B peptide (this eliminates antibody-mediated neutralization) The infectivity of the virus was detected. More recently, the use of 16B antiserum Immunocytochemistry studies detected the antigen in FLY-infected cells. 16Bpe Blocking this antiserum with peptide (which interferes with antibody recognition of the antigen) .

Increased cell coloration in an apparently non-specific manner.

plsE (denoted as ISP) and the amino acid sequence LQNRRGL Other novel peptides with DI LFLQEGGLC (denoted as ISP) shows immunosuppressive activity.

The present invention binds to an ISP domain and has the following amino acid sequence. An immunosuppressive polypeptide consisting of the 16B portion, designated as 816B': ISP to provide: AKLRERLKQRQQLQNRRGLDI LFLQEGGLcl-1-δ 16B--11--------I 5P-----lδ16B: ISP A soluble polypeptide that inhibits in vitro mitogen-induced differentiation of Zumitile T lymphocytes. be. The observed growth inhibition cannot be assumed to be a function of cytotoxicity. Also, live Other peptide configurations that are active include heptad, shimmer, and multimeric forms. Related sequences from retroviral and cellular proteins, inverted structures ( ISP:16B), cut the tip? =16B:ISP and ISP:16B Pepti includes the code. Beyond carrier molecule-bound peptides; the advantages of these peptides are High solubility, low immunogenicity and biological activity at lower (W/V) concentrations includes.

The present invention also has a molecular weight of about 110,000 daltons, and to purified polypeptides detected on cancer cells. This polypep Tide is an immunosuppressive protein derived from the predicted amino acid sequence of feline leukemia virus p15E. Detected with antiserum raised against the polypeptide. This 110,000 Dal Antibodies that detect tons of peptides can be used in the diagnosis of many malignant diseases, and in the treatment of remission. It is useful for monitoring the peripheral blood of patients with hematological disorders for residual disease. So Additionally, this polypeptide plays a role in immune leukemia and tumor cell-mediated destruction of cancer. a synthetic peptide or its receptor, as it plays a functional role; as well as synthetic peptides derived from its predicted amino acid sequence. t; has therapeutic use for modulating tumor immunity.

For cats, human T cells and feline leukemia virus inhibitory peptide (ISP) Raised polyclonal rabbit antibodies were generated and affinity purified to F LY-ISP antibody was obtained. Immunocytochemical analysis using such FLY-ISP antibody The coloring strongly detects the antigen in FLV infected feline cells. However, ISP anti- The body also detects antigens in various cell lines established from human leukemia patients. However, it is not detected in normal murine fibroblasts. of peripheral blood from a normal individual. Antibody staining of white blood cells (PBL) is based on leukemia cells or phytohemagglutinin (1 123 bulb mitogen) stimulation is very weak compared to PELs. [! 55] Methionine Immunoprecipitation from FL74 metabolically labeled for 4 hours at gp85 and a molecular weight of approximately 110,000 Daltons. Ripeptide was detected. Similar experiments using the human leukemia cell line RajiW have shown that approximately 35 ．． 000 (35K), 37.000 (37K) and 110.000 (l l0K) Three types of proteins with a molecular weight of Dalton were detected. ll0k egg White matter was detected in human leukemia cell lines 562, EM2 and HL60; , 35 and 37 were not detected. ll0K is also a human epidermal carcinoma cell in line A431, but not in normal murine 3T3 fibroblasts. I couldn't. Using centrifugation speeds that precipitate virus-associated p15E, human white The ll0K protein from hematopoietic cells was extracted from the supernatant of the conditioned growth medium. detected in the image. This soluble 110 protein is present in various leukemias and solid tumors. released into the growth medium by a cell line established from Epitopes that are structurally and functionally similar to the domain of the peptidergic domain are shown.

Ba1b/c tile cells were treated with murine T cell mitogen ConA (4 μg/ml) 8 and a titrated amount of the δ16B:ISP peptide for 4 days. δ16B: Jjh exposure to rSP peptide; differentiation of mitogen-stimulated cultures cells and mitogens only) expressed as a percentage. The dashed line represents the positive control culture (thin This shows the differentiation of cells + mitogens only).

Ba1b/c tile cells were incubated with ConA and titrated amounts of these peptides for 4 Cultured for 1 day. Dashed line indicates differentiation of positive control culture (on-cell mitogen only) .

111-16B ・-・Lu ISP Mumufu δ16B: ISP Figure 3. δ16B: TSP-induced epidemic-suppressing Ba1b/c tile cells were transferred to ConA (A ) or stimulated with LPS (B) in the presence of a titrated amount of 815B:ISP peptide. . Cell cultures were incubated for 4 or 5 days. (A) T cell differentiation Effect on. (B) Effect on B cell differentiation.

Mumu 4th day ・-・Iris 5th day Figure 4. Suppression of lymphocyte proliferation Ba1b/c tile cells were cultured in the presence of ConA and a titrated amount of the 816B:ISP peptide. 3.4 mats were cultured for 5 days before harvest. Proliferation values are for control cultures (cells and and ConA only).

・－・－3rd day ■－■－Day 4 Moom = 5th day Bind the δ16B:ISP peptide to microtiter wells and add standard Different peptidophilic antibody preparations in enzyme-linked immunosorbent assay (ELISA) At the same time, I made an ink bath scene. AP448-28, against ISP peptide Affinity purified rabbit antiserum; mouse against 9-14F2-3,1SP peptide Monoclonal antibody, rabbit antiserum against R112-5,16B peptide; A P67, an affinity-purified rabbit antiserum against the c-abl gene product (negative control).

・-・-σ-I SP (AP55-28) Mumu-a-ISP (9-14F 2-3)-1■=a-16B (R112-5)0-0-a-abl (A P.67) A detailed description of the present invention and specific embodiments is included within the scope of this application. Diamino E1 3 letters 1 letter listing amino acids and abbreviations used L-tyrosine tyr Y Glycine G L-phenylalanine phe F L-methionine met M L-alanine ala A L-serine ser S L-inleucine iso I L-leucine leu L L-threonine thr T L-valine val V L-proline pro P L-lysine 17s K L-histidine hisH L-glutamine gin Q L-glutamic acid glu E L-tryptophan trp W L-arginine ar(R L-asparagine asp D L-asparagine asn N L-cystine cys C Not specified The present invention comprises peptides having less than about 40 amino acid residues, The amino acid residue consists of an amino acid sequence of at least five amino acid residues, and The acid sequence is the amino acid sequence LQNRRGLDILFLQEGGLCAALKEECC Provides an immunosuppressive peptide selected from amino acid sequences included within F. Ru. Within the scope of this application, a "peptide" has from 2 to about 100 amino acids. Peptide refers to a chain of amino acid residues and is purified from naturally occurring products. or produced by synthetic or recombinant DNA methods; Contains. Amino acid chains having greater than about 100 amino acid residues are herein referred to as polypeptides. It's called a peptide. In one embodiment of the invention, the peptide is conjugated to a carrier molecule. to form immunosuppressive compounds. Within the scope of this application, "carrier molecule" means Conjugating, e.g. cross-linking, to the ligand in question increases the immunogenicity of the ligand or molecules that activate the biological activity of the ligand, e.g., immunosuppressive activity. means. Carrier molecules that increase the immunogenicity of a ligand are known in the art. and large proteins, such as keyhole limpet hemocyanin (KL H), ovalpumin, butyroglobulin and bovine serum albumin (BSA) , lipids, and peptides. Carrier component that activates the biological activity of the ligand Examples are also known in the art, such as polyethylene glycol It is. Methods for conjugating peptides to carrier molecules are known in the art. , and in: Lerner et al., PNAS (USA) (1981) Eiro: 3403-3407, Chureh ) et al., PNAS (USA) (1983) 80: 250, and Erlanga -(Erlanger) Methods of Engineering f　Enzy+++ology)　(1980)ヱ0:85-104e peptide : Coupling agents useful for preparing carrier molecule conjugates are known in the art. Known and customary crosslinking agents, such as aldehydes, such as glutamate aldehyde, carposiimide, succinimide, bis-diazotized benzazine, and imidate. Applicant also discloses that certain amino acids, viz. Stine or lysine at the carboxy or amino terminus of the peptides of the invention. Consider adding it to the carrier molecule for the purpose of crosslinking. Preferred implementation of the invention In embodiments, the carrier molecule is keyhole limpet hemocyanin. of the present invention In other embodiments, the carrier molecule is attached to the peptide using glutaraldehyde. match.

The present invention also provides peptides comprising less than about 40 amino acid residues, The amino acid residues described above consist of an amino acid sequence of at least 5 amino acid residues, and The amino acid sequence is LQNRRGLDILFLQEGGLC.

A-ALKEECCFLKEEC.

QEGGLCAALKEEC.

KSLTSLSEVVLQNRRG.

LQARI LAVERYLKDQQLlAVERYLKDQQLLG IWG C3GKL IC.

QREKRAVG I GALFLGFLG.

QLTVWG I KQLQARI L.

LQNRRGLDLLLFLKERGLC.

AQNRRGLDLLFWEQGGLC.

LQNRRGLDLLTAEQGG IC.

AQNRRGLDWLY I RLGFQSlAKLRERLKQRQQ.

LRNRRAL I LLAQMGRIS.

LDNRRTLMLLAQMSRIS.

LGNRRAL I LLAQMRRI 51LGNRRAL I LLGQM GRI S.

LNNRRTLMLMAQMRRIS.

PVNPR5LEKLE, I IPASQ, OJ: and LGSRRTLMLLA QMRK IS.

amino acids included within the range of the amino acid sequence selected from the group of amino acid sequences consisting of an immunosuppressant selected from amino acid sequences, said peptide being conjugated to a carrier molecule; provide a compound.

The invention still further provides a method comprising peptides having less than about 40 amino acid residues. and the amino acid residues consist of an amino acid sequence of at least 5 amino acid residues. , the amino acid sequence is the amino acid sequence QREKRAVGIGALFLGFG or Amino acid sequence included within the amino acid sequence QLTVWGIKQLQARIL Provided are immunosuppressive peptides selected from: Furthermore, other immunosuppressive peptides have been proposed. and this peptide has the amino acid sequence LQNRRGLDILFLQEGGLC consists of antigenic determinants homologous to those of the peptide having This application Within the range of antigenic determinants, antigenic determinants can be defined as homology to other antigenic determinants.

Furthermore, the present invention provides a first amino acid sequence of at least five amino acid residues, The first amino acid sequence is included within the amino acid sequence AKLRERLKQRQQ. and at least five amino acid residues selected from the amino acid sequence and one other amino acid sequence, the amino acid sequence of said ability is LQNRRGLDI LFLQEGGLC.

AALKEECCFLKEEC.

QEGGLCAALKEEC.

KSLTSLSEVVLQNRRG.

LQARI LAVERYLKDQQL.

AVERYLKDQQLLGIWGC5GKL IC.

LQNRRGLDLLLFLKERGLC.

AQNRRGLDLLFWEQGGLC.

LQNRRGLDLLLTAEQGGIC.

AQNRRGLDWLYI RLGFQS.

LRNRRAL I LLAQMGRIS.

LDNRRTLMLLAQMSRIS.

LGNRRAL I LLAQMRRIS.

LGNRRAL I LLGQMGRIS.

LNNRRTLMLMAQMRRI 51PVNPR5LEKLE I I P ASQ.

LGSRRTLMLLAQMRK I S.

QREKRAVGIGALFLGFLG, and QLTVWGIKQLQARI L.

amino acids included within the range of the amino acid sequence selected from the group of amino acid sequences consisting of Provided are immunosuppressive compounds selected from amino acid sequences.

Peptides provided by the invention can be cyclic or polymeric. or shimmer units.

In one embodiment of the invention, within the amino acid sequence AKLRERLKQRQQ The amine terminus of the included amino acid sequence is LQNRRGLDILFLQEGGL C.

AALKEECCFLKEEC.

QEGGLCAALKEEC.

KSLTSLSEVVLQNRRG.

LQARILAVERYLKDQQL.

AVERYLKDQQLLGIWGCSGKLIC.

LQNRRGLDLLLFLKERGLC.

AQNRRGLDLLFWEQGGLC.

LQNRRGLDLLLTAEQGGIC.

AQNRRGLDWLYIRLGFQS.

LRNRRALI　LLAQMGRI　S.

LDNRRTLMLLAQMSRIS.

LGNRRAL I LLAQMRRIS.

LGNRRAL I LLGQMGRIS.

LNNRRTLMLMAQMRRIS.

PVNPR5LEKLE I I PASQ.

LGSRRTLMLLAQMRK I S.

QREKRAVGIGALFLGFLG, and QLTVWG I KQLQA RI L.

amino acids included within the range of the amino acid sequence selected from the group of amino acid sequences consisting of It is attached to the carboxy terminus of the amino acid sequence.

In another embodiment of the invention, within the amino acid sequence AKLRERLKQRQQ, The carboxy terminus of the amino acid sequence contained is LQNRRGLDILFLQEGG L.C.

AALKEECCFLKEEC.

QEGGLCAALKEEC.

KSLTSLSEVVLQNRRG.

LQARILAVERYLKDQQL.

AVERYLKDQQLLGIWGCSGKLIC.

LQNRRGLDLLLFLKERGLC.

AQNRRGLDLLFWEQGGLC.

LQNRRGLDLLLTAEQGGIC.

AQNRRGLDWLYIRLGFQS.

LRNRRALLILLAQMGRIS.

L,DNRRTLMLLAQMSRIS.

LGNRRALILLAQMRRIS.

LGNRRAL I LLGQMGRIS.

LNNRRTLMLMAQMRRIS.

PVNPR5LEKLE I I PASQ.

LGSRRTLMLLAQMRK I S.

QREKRAVGIGALFLGFLG, and QLTVWG I KQLQ ARI L.

amino acids included within the range of the amino acid sequence selected from the group of amino acid sequences consisting of It is attached to the amino terminus of a amino acid sequence.

In yet another embodiment of the invention, the amino acid sequence AKLR-ERLKQRQQ Each of the carboxy-terminus and amino-terminus of the amino acid sequence contained within LQNRRGLDILFLQEGGLC.

AALKEECCFLKEEC.

QEGGLCAALKEEC.

KSLTSLSEVVLQNRRG.

LQARILAVERYLKDQQL.

AVERYLKDQQLLGIWGCSGKLIC.

LQNRRGLDLLLFLKERGLC.

AQNRRGLDLLFWEQGGLC.

LQNRRGLDLLTAEQGGIClAQNRRGLDWLYIRLGFQ S.

LRNRRALILLAQMGRIS.

LDNRRTLMLLAQMSRIS.

LGNRRALILLAQMRRIS.

LGNRRAL I LLGQMGRIS.

LNNRRTLMLMAQMRRIS.

PVNPR3LEKLE I I PASQ.

LGSRRTLMLLAQMRK I S.

QREKRAVGIGALFLGFLG, oh! UQLTVWG I KQLQ ARI L.

amino acids included within the range of the amino acid sequence selected from the group of amino acid sequences consisting of It is bound to a amino acid sequence.

An amino acid attached to the carboxy terminus of an amino acid sequence encompassed within the amino acid sequence. The amino acid sequence is the amino acid sequence encompassed within the amino acid sequence AKLI'1ERLKQRQQ. can be the same as or different from the amino acid sequence attached to the amino acid sequence 0 In a preferred embodiment of the invention, the immunosuppressive peptide is AKLRERLKQ 9 Other preferred embodiments of the present invention consisting of RQQLQNRRGLDILFLQEGGLC In embodiments, the immunosuppressive peptide is AKLRELKQRQQLQNRRGL Consists of DILFLQEGGLC.

The present invention also has the amino acid sequence LQNRRGLDILFLQEGGLC antigenic determinant for which antibodies raised against an amino acid sequence have affinity V4-produced polypeptides consisting of V4 are provided. Here, the polypeptide is Expressed in mammalian cancer cells. Within this application, An embodiment of the invention refers to an antibody capable of binding to an antigenic determinant. In, M! ! ! The resulting polypeptide has an apparent value of approximately 110,000 Daltons. In other embodiments of the invention, the purified polypeptide has a molecular weight of about It has an apparent molecular weight of 35,000 Daltons.

Additionally, each of the purified polypeptides provided herein is encoded (e n c o d e) A purified nucleic acid sequence is provided. Such a nucleic acid sequence , DNA, RNA and cDNA sequences. This is useful for preparing expression vectors capable of expressing the vector. Furthermore, refined One of the invention provides antibodies having affinity for each of the polypeptides. In other embodiments of the invention, the antibody is a polyclonal antibody. In this case, the antibodies are seven clonal antibodies.

Furthermore, one aspect of the present invention provides that the present invention has an affinity for purified polypeptides of the present invention. Provides purified protein. This purified protein.

Present on the surface of hematopoietic cells.

A therapeutic composition comprising an antibody of the invention and a pharmaceutically acceptable carrier may also be used. , provided. The therapeutic composition provides a therapeutically effective immunosuppressive blocking amount to the subject. By administering a therapeutic composition, the subject is immunologically or hematopoietically suppressed. Other therapeutic compositions useful in methods of treating subjects with is provided. This therapeutic composition comprises one purified polypeptide of the invention, and is a peptide having an amino acid sequence of at least 5 amino acids; sequence is contained within said purified polypeptide and is pharmaceutically acceptable. It consists of a carrier. The therapeutic composition provides an effective immunosuppressive amount of the therapeutic composition to the subject. It is useful in methods of suppressing a subject's immune system by administering the compound.

Still further, one of the purified proteins of the present invention having immunosuppressive peptide binding activity and a pharmaceutically acceptable carrier. child The therapeutic composition comprises administering to a subject an effective immunosuppressive blocking amount of the therapeutic composition. How to treat immunologically or hematopoietically suppressed subjects by Useful in law.

A method of detecting cancer cells in a sample, such as a whole blood sample or a bone marrow sample, is provided, This method uses a peptide having the amino acid sequence LQNRRGLDILFLQEGGLC. develop a polypeptide with antigenic determinants that bind to antibodies raised against detecting from a sample cells expressing a mammalian cancer. Expressed in cells.

Additionally, a method of diagnosing cancer in a subject is provided, the method comprising: In the collected body fluid sample, the amino acid sequence LQNRRGLDILFLQEGG A peptide having LC or a part of the polypeptide containing the antigenic determinant Detects polypeptides with antigenic determinants that bind to antibodies raised against and the polypeptide is expressed in mammalian cancer cells. .

Still further, from an immunosuppressive peptide of the invention and a pharmaceutically acceptable carrier, A vaccine will be provided. This vaccine must be administered to the subject at an effective immunizing dose. to immunize the subject against retroviral infection by administering It is useful for

Yet still other vaccines are provided, which include the immunosuppressive compounds of the present invention and and a pharmaceutically acceptable carrier. This vaccine provides effective immunity to the subject. By administering a large amount of vaccine, subjects can be protected against retroviral infection. Useful for immunization.

The present invention further provides immunosuppressive peptides of the present invention and pharmaceutically acceptable carriers. A therapeutic composition comprising the body is provided. This therapeutic composition.

immunization of a subject by administering to the subject an effective immunosuppressive amount of the therapeutic composition. useful for suppressing the system.

Finally, one treatment comprising an immunosuppressive compound of the present invention and a pharmaceutically acceptable carrier. A therapeutic composition is provided. The therapeutic composition provides a therapeutically effective immunosuppressive amount to the subject. Administration of the composition is useful for suppressing a subject's immune system.

Applicant has disclosed that synthetically produced and keyhole limpet hemocyanin (KLH) ISP cross-linked with glutaraldehyde suppresses Midoken-induced mT cells in vitro. However, KLH alone showed no immunosuppressive activity. Antiolo (Ci When using the peptide reported by AncioJo), ISP When not conjugated to offspring, it was not immunosuppressive.

Synthetic ISP cross-linked to KLH with glutaraldehyde was also synthesized by Freund's adjuvant rabbit polyclonate using standard immunization techniques. null antiserum was generated. This antiserum was used as an immobilized antibody against ISP and peroxidase. A Sidase Ia:a-loving rabbit antibody is used. For standard ELASA immunoassays was observed to specifically recognize ISP.

Unexpectedly, the antiserum was also analyzed by standard Western blot. , FLV R15E and its precursor polyprotein (gPr 85 env) was observed. This was surprising.

This is because antibodies to peptides often do not recognize the natural protein or Or, if they were recognized, they were only weakly recognized. peptide parent Immunocytochemical analysis using purified antibodies revealed that antibodies were detected in FLY-infected cells. The specificity of the antibody for the origin was confirmed. Because the antibody together with ISP in solution incubation blocks antibody reactivity with p15E-related proteins. This is because it was found that the

Using a peptide affinity-purified rabbit antibody against ISP, Western C. F+, clearly or exogenously infected by a retrovirus, by test analysis. protein antibodies derived from cell lysates of several human leukemia cell lines. Screened. This study included 562. EM2. CEM and Raji human Leukemia cells were included. Surprisingly, antibodies also target each of these cells. A protein or proteins having an apparent molecular weight of about 35,000 Daltons. and specifically identified them. This protein is designated as p35. normal White blood cells and normal fibroblasts in human peripheral blood contain this protein or proteins. , and thus did not provide a binding site for anti-peptide antibodies.

Thus, antibodies against ISP of the present invention can be used to target P15E-related proteins in human leukemia cells. In order to detect the white matter, and in which the protein for P15E is expressed, a variety of Useful for diagnosing naturally occurring human cancers. Additionally, retroviruses Infected cells also express P15E-related protein 1, such as FLY-infected cells. Antibodies to ISP or related sequences may contain such viruses. for detecting viral antigens in tissues or fluids, and for retroviral rus-induced diseases, such as Hxva-induced AIDS, FLY-induced A:i leukemia, B LV: fi-11 bovine leukemia, and induced by endogenous retroviral sequences. It is useful for diagnosing other as-yet-unknown diseases.

P35 protein reacts with antiserum against retrovirus p15E and ISP Based on the antigenic homology between p35 and p15E, p35 is an immunosuppressive protein, similar to p15E. or a set of related proteins. Immunosuppression and p35 The link between is 562 for research on human tumor cell lines. It can be estimated from research. In 562 cells, red-white blood cell plast crisis ( blast (crisiS) derived from chronic myeloid leukemia (CML) Ta. The established cell line is characterized by the expression of an oncogene called bar-abl. I get kicked. Expression of this gene was expressed in Philadelphia (Philadelphia). a) Resulting in the formation of stray chromosomes called chromosomes. Interchromosomal inheritance in patients This is the result of exchanging child information. This altered chromosome formation.

Expression of a hybrid gene composed of so-called bcr and abl sequences will occur. Expression of this gene is chronic, as the abl gene is a known oncogene. It is thought to cause myeloid leukemia.

An anti-Ik1 serum directed against the bcr and abl proteins was released [Bl] and b cr-abl hybrid gene product (P 2 t o b Cr-&b 1 or P210) was used to identify these in 562 cells. [Nature 315:550.1985], P210 was , its ability to autophosphorylate while in its antibody complexes (immune complexes) can be detected by Certain drugs, such as Folpar 12-Iristi) 1 When 620 cells were treated with 3acetate (PMA) and mezerein, P2 of activated oncogene P210 such that intracellular expression of P210 is dramatically reduced. Changes in expression are induced. Treatment with these factors also increased the number of cells in 562 cells. transformation and loss of the transformed phenotype.

As mentioned above, 562 was detected by detection with the anti-ISP antibody of the present invention. It contains p35, as shown in FIG. It is one of the human cell lines.

In addition to the effects of P210 on aquatic organisms, treatment of Ni562 cells with PMA , also causes a dramatic decrease in the levels of p35; thus, p35 expression is reduced in CM Directly correlated with expression of the L oncogene P210. As mentioned above, to 562 cells and other established cell lines derived from human leukemia patients are Inhibits in vitro growth [Olofson (01ofsssoon) and Olofson Son (015son), Leukemia REs 65:437.1980; and 8:387.1984; Steiberg et al., Bloo d 65:100.1985] and inhibiting mitogen-induced differentiation of peripheral lymphocytes. [Chiao et al., PNAS 83:3423, 1986] Some It is known to secrete factors such as Moreover, the expression of these suppressed inner children is In 20, inducing differentiation with hemin was shown to decrease. These consequences As shown in the results, P35 is an inhibitor previously described to be secreted by 562 cells. P35 is a regulatory factor and is involved in disease states of 562 cells. p A direct correlation between the expression of P210 and the enzymatic activity of P210 also shows that CML EM [W, Kretosar (K l o e t z e r ), unpublished results], both P210 and p35 levels in this cell line. cells expand upon treatment with PMA, and cells resolve their transformed phenotype. EM2 cells are derived from patients with myeloid plastophysis.

These findings suggest that CML cells of different cell lineages respond differently to PMA. However, the results also suggest that there is a relationship between oncogene expression and p35 expression. Establish a correlation.

Based on the correlation between p35 water products and the expression of activated oncogenes, p35 against p35, representing a target for the development of new therapeutics or vaccines. antibodies against p15E or immunosuppressive peptides derived from p15E or P35 sequences. Therefore, these antibodies in human tumors and retrovirus-induced diseases are important. It is useful when blocking the inhibitory effect of p15E-related proteins. or 1 Synthetic peptide analogs may inhibit the inhibitory effects of inhibitory proteins. Such analogs compete for receptors of immunosuppressive peptides, thereby increasing immunity. They block the binding of immunosuppressive proteins, but they themselves act as a trigger for immunosuppressive events. It is biologically inert because it cannot initiate a sequence.

Immunosuppressive p15E-related as a vaccine for prevention of retrovirus-induced diseases To illustrate the value of the peptide, applicants conjugated it to KLH with glutaraldehyde. and formulated with Freund's adjuvant. Immunized four cats with ISr. Ta. Three of the four cats were protected from viremia when challenged with FLY. However, 6 of the 8 controls became viremic. As these results show In addition, ISP may be useful as an effective vaccine in the prevention of FLY viremia. Ru.

Applicants are further able to suppress mitogen-induced differentiation of T cells in vitro. three pe The peptides were derived from HIV sequences. Amino acid sequence QREKRAVGIGALFL One peptide with GFG (designated SUP2) is found in the HIV major envelope. transmembrane glycoprotein gP120 and transmembrane glycoprotein gp41 (amino acids 514-53 1) spans the joint. This peptide shows a small degree of homology with ISP. Other immunosuppressive compounds having the amino acid sequence LQARILAVERYLKDQQL (denoted as SUPl) was added to the gp41 region of HIV (amino acids 583-5 99) and had no significant homology to ISP.

Both 5UPI and 5UP2 respond to mitogenic stimulation when conjugated to Kl,H. and suppress T cell differentiation. However, unexpectedly, 5UP2 , also without conjugation, is immunosuppressive.

Other immunosuppressive peptides (designated 34,1 and the amino acid sequence AYERYLK DQQLLGIWGC5GKLIC) was derived from gp41, and Although it has no homology to ISP or 5UP2.

Overlaps with the 5UP1 sequence. 34.1 also when joined to KLH.

Alternatively, when used unconjugated, it has been shown to be immunosuppressive in vitro.

Applicants believe that these peptides may be used as therapeutic immunosuppressants to prevent HIB infection. and its usefulness as a vaccine. These HIV immunosuppressive drugs Antibodies (monoclonal or polyclonal antibodies) against AI It is expected to have diagnostic and therapeutic potential in DS.

The peptides, polypeptides, proteins, compounds and methods of the present invention can be used in the following experiments and methods. These may be better understood by reference to the description and examples. The experiments and examples are for illustrative purposes only and are defined by the claims. should not be construed as limiting the scope of the invention in any way.

Group 1 Gotota Example 1 - Preparation of peptide-keyhole limpet hemocyanin (KHL) conjugate made Peptides were synthesized by conventional solid-phase methods and cross-linked to KLH in the following manner. ), peptide (5 mg/ml in water) and KLH (5 mg/ml in water) 1) were mixed together. Gulguraldehyde was added to this mixture at 0.04% (v /v). The reaction mixture was stirred at room temperature for 30 minutes and then Dialysis was performed overnight at 4°C against phosphate buffered saline (PBS).

Example 2 - Antibody generation and purification New Sealant: Add 0.50 ml of PBS and 0.05 ml of complete 200 pg of conjugate in Freund's adjuvant was injected. incomplete freund 200 in adjuvant. Two booster injections of gtR combination/PBS for 4 weeks. Administered at intervals. Every week, a sample of nn supernatant was analyzed with immobilized synthetic peptides by ELISA. Recognizing 0 then 1 synthetic peptides monitored for titers of antibodies against Serum antibody samples with known More complete antigen recognition was tested. In this test, FLY-infected cells ( The extract of cell line FL74) was resolved by SDS slab gel electrophoresis and Electrophoretically transferred to nitrocellulose membrane. Tris I containing 3% gelatin II! After blocking with #1 solution, swipe the membrane vertically onto the same strip. I had rice. These split cell protein strips.

P15E synthetic peptide diluted in Tris-buffered saline containing 1% gelatin. probed with antiserum against Tide and peroxidase-labeled rabbit Ig. . Final visualization of antibody reactivity was performed in an enzyme substrate solution of 4-chloro-1-naphthol. This was done by incubating the strips with virus p1 All of the serum samples that strongly recognize 5E were pooled and EL was applied to the peptide. Powered by ISA and by immunoplot studies against whole (p15E) antibodies. The price has been decided. In all immunoplot studies, nonspecific antibody staining is Identical plot with antiserum blocked with non-binding peptide from antibody detection were easily distinguished by probing.

determined in peptide blocking experiments. Immunocytochemical detection of antigens is , obtained affinity purification of antibodies. Rabbit anti-peptide antibody was prepared using CM-774 gel block. Pass the antiserum through an Affigel-Blue (Bio-Rad) column. Purification was performed to obtain an enriched Ig fraction. This Ig fraction was Oxyactivated Sepharose 4B-? It was applied to the prepared peptide affinity column. this The column was washed with 10.5 molar KCI in phosphate buffer and the antibody was washed with 3 molar KCI. of phosphate buffer.

Elution was performed by placing 1 molar acetic acid in a tube containing pH 8. active fraction Pooled, dialyzed, and concentrated by ultrafiltration. All steps of antibody enrichment Progress was monitored by peptide ELISA. Immunocytochemistry procedure is a European patent Fully labeled secondary antibody and silver enhancement as described in Publication No. 158,746. It was used.

Example 3 - gl of samples for immunoplot analysis! ! ! Aliphatic immunoplot Analysis revealed that rA was made by first rinsing in PBS and then lyophilization. Ta. The resulting cell powder (6-Omg/m1) was suspended in SDS sample buffer. , and further denatured in rapidly boiling water. Clean the cell lysate at room temperature. (3Q, QOOrpm: Be7kuman 50 Ti rotor), 200. from g The extract was subjected to sodium dodecyl sulfate polyacrylic gel electrophoresis (SDS-P AGE) using a 3% stacked 712% resolving gel. protein electrophoretically, 192 mmol glycine/20% methanol/10 mole Mol Tris, 3 hours at 100 volts and overnight at 40 volts in pH 8.3. Transferred to trocellulose membrane.

Example 4 - Virus Il! by Western blot analysis in extracts of tumor cell lines. Comparison of antiserum detection of antibodies Rabbit antiserum against non-inhibitory feline p15E peptide and ISP.

Immunoplot tests were performed for the detection of antibodies in virus-infected cell extracts. intact Goat polyclonal antisera against murine p30gag and gp70env were , FL74 (FLY infected Linse II! induced cell line) and NRK206-2/ in IC (murine leukemia/melanoma virus [MLv/SV] infected) cells. The existence of virus proteins was confirmed. Both antisera against p15E synthetic peptide were , Pr85elv, p15E and P12E in FL74 cells (proteolytically processed p15E) was detected, but MLV/SV or adult (adult) No T-cell leukemia virus ()ITLV I) homolog was detected. anti-ISP blood The supernatant detected the protein (p35) of HT but not the other two cell lines. Ta.

Using anti-ISP, purified PBLs and species derived primarily from human acute leukemia Extracts of each cell line were immunoplotted (Table 1) for all leukemia cells tested. The system expressed P35 protein to the exclusion of normal PBLs. Antiserum reaction with antigen Responsiveness can be blocked in all cell lines with soluble, unconjugated peptides. King was king. The highest level of protein expression in 35 was found in the Raji cell line. was detected.

Table 1 Cell antigen anti-ISP serum detection book Immunoplot analysis Cell p35 immunocytochemistry FL74 Pr85env, p15E +++Raji + +++ +EM 10 +++ HUT102 + +++ HL60 + +++ 562 + +++ EM2 + +++ Human PBLs 10 A4318 + ++ Antiserum detection of antigens blocked with peptides without wood complex binding; thus, (− ) indicates that the antigen is not detected: (+) = weak; (++) = moderate; and (+++) = strong.

Upon exposure to C kinase activator, the activated oncoprotein P210ber-ab A change in the expression of Kl is induced [Klenzer et al. , unpublished results], an immunocytochemical comparison of induced versus uninduced cells. , established a direct correlation between the expression of P210bcr-abl and P35. (Table 2), for this reason Applicants believe that expression of p15E-related proteins regulates differentiation. Insist that an event occur. The variation in the amount of P35 detected is a result of changes in the rate of synthesis. or whether it is the result of altered processing of the detected precursor protein. It has not yet been determined.

Table 5 PMA exposure altered protein expression in 35 cells 10 nM PMA P21 0bcr-abl p35 has no 562 ++++ 562 5th day -- EM2 None + + EM2 5th 10++++ P210bcr-abl levels were determined by in vitro kinase assay.

No protein detection in P210bcr-abl and 35 -), weak (+) , tested as moderate (++) or strong (10+).

Example 5 - Definition of tumor epitopes recognized by anti-ISP serum , Pr85env, p15E and P12E in FLY-infected cells, and testing specifically detected the 35,000 Dalton cell antigen in all human leukemia cell lines tested. I recognized it. I#, the specificity of anti-ISP serum for wound proteins is homologous, That is, proteins synthesized from murine, human, and bovine mountain rot virus env sequences. Tested by blocking antiserum, similar but not identical to peptide The ability to repeat 0 duplicates was tested. The results of this test were The antibodies bound to the white matter are primarily directed against the variable portion of the cat ISP. and

Table 3 Peptide of anti-ISP serum recognition of viral pile 1 (FL74) and cellular antigen tree blocking Blocking sequence Pr85env/p15E' p35 QEGGLCAAL KEEC÷÷ LDILFLQEGGLCAALK (ISP) LQNRRGLDILFLQEGGLCEVVLONRRRGLDI LFL + +KSLTSLSEVVLQNRRC+ + (MLV) LQIJ RRGLDLLFKEGGLC (HTLVI/2) AQNRRGDLLFW EQGGLc + + (BLV) AQNRRGLDWLYIRIGFQS + +YONRLALDYLLAAE (, GVC (endogenous human virus) *antigen Detection was evaluated as positive (+) or negative (-): endogenous human virus sequences was not tested.

The viral p15H ISP domain is the most sophisticated of the RNA tumor viruses. Saved in! : Peptide domain. This storage is shown below (Table 4) in FLV and and predicted amino acid sequences for domains of the HTLV1 envelope (sequence determination). Alignment (a I i gnnoe nt) (knee identical match).

Table 4 FRQLQilAIIHTDIQ LEEISALEKSL ding 5LSE%°% LQNRRGLDILFLQEGGLCAALKEECCFP^DHTG GKSLL) IEt'DK DISQLTO, I%' KNH LLKIA QYAAQNRRGL DLLFIEQ to GL CKALQd RF PNIT N.S. HTLI°1352 362 372 3g2 392 402 significant homology Only one region is known by John Elder, who calls it Cl8B. [Scripps In5titut3 Personal Communiqué] ISP peptides and neutralizing antibodies (e.g. For example, antibodies that block in vitro virus infection) are present almost in the center.

Computer alignment can be done using similar hydropathic techniques. ``matches'' If performed, excluding scoring with J, the following alignment is achieved: (Table 5).

Equivalence of the same matching decimal order: Neutral or weakly hydrophilic: A-G-P-3-T Hydrophilic with small side groups: D- N-E-Q hydrophilic with large lateral groups: hydrophilic with small lateral groups in R-H- Aqueous: D-N-E-Q Hydrophobic with large side groups: R-H- to 1... --ISP---------・----(FLY l 540 5501 560K NHKNLIJIA QYAAQNRRGL DLLFWEQGGL CKAL QEQCRF PNITNSHVHTL%' 1372 382 392 40 2 Although exact sequence homology is not preserved in the C-terminal half of the Summer SP domain, , it is clear that tree parentage is highly conserved. For this reason, the applicant The amino acid sequence of the end portion and possibly FLY amino acid 560 is p15E immune. We believe that it plays an important role in inhibitory activity.

The overall homology between the envelope sequences of FLV and HTLVI or 2 is In the SP domain, it is slight but very clear.

We also investigated the relationship between FLY and human immunodeficiency virus (HIV) envelope genes. Very clearly related sequence homologies between the gene products were identified (Table 6).

Table 6 <-----Nol5r--------1in FLV PI5E 56-103 63 5LSE%’V LQNRRGLDILFLQEcGLCAALKEECCn ’ADHT 100 books Nishiki 0 Korea 0 Ne 0 0 0 0 0 0 0 0 books: is the same match show.

* indicates identical match and equality of: A-G=P-5=T F=W=Y 1-L-M-V Q-D-E=N K=)l=R Example 6 - Immunosuppression caused by viral p15E and p15E-like proteins antibody blocking of Persistent infection with feline leukemia virus results from suppressed cell-mediated immunity , often associated with non-regenerative aplastic anemia and opportunistic infections. these The clinical features have been attributed to the direct action of viral p15E. Rat p15 Biologically active synthetic peptides selected from E amino acid sequences have been developed by other researchers. demonstrated that it exhibits effects similar to intact viral proteins in vitro. I;. Applicant has proposed that a synthetic peptide from the homologous region of feline virus p15E It was confirmed that it has a positive effect. Applicants also believe that the ISP:KLH conjugate Feline virus p15E and many others, as determined by body peptide blocking. specifically recognized cross-reactive antigens in human leukemia cells.

Peptide blocking experiments using overlapping sequences are directed against the antibodies; shows that the major nipitope is within the carboxy-terminal half of the ISP sequence. t;. Kohler and Milstein ( ISP generated using the basic method of Nature 225.1975). A monoclonal antibody directed to the (Table 3).

Another approach is to generate purified monoclonal antibodies against cat 15, And hybridoma clones! sP's E L I S A recognition 111: Just screening. The unique advantage of this approach is that natural protein development of monoclonal antibodies directed against biologically active sites on the protein. antigen After preliminary selection of clones for recognition of screened for the ability to block virus-mediated inhibition of tile cells can do. Inhibition of viral p15E by binding to the active site Monoclonal antibodies that block activity reduce symptoms of virus-induced diseases 0 pl detected in various human cancers can be tested for the ability to Similar studies of effects on sE-like proteins may also be used as passive immunotherapeutic agents. Alternatively, it can be tested as another drug that labels cancer cells.

Example 7 - Conserved synthetic peptide virus p15E as an immunosuppressive therapeutic agent domain (all FLY domains listed in Table 5) or derived from plsE-like proteins. Synthetic or recombinantly (DNA) derived peptides with amino acid sequences that Can act as an inhibitor. Such peptides are useful for tissue or organ transfer. Useful for suppressing the immune system after transplantation. Additionally, biologically active peptides can provide therapeutic benefit to patients with autoimmune diseases.

Example 8 - Synthetic peptide feline IsP as a retrovirus vaccine: KLH The conjugate was used to immunize four cats. 200μ in 0°5ml DPGS g of conjugate + 1.g of conjugate in 0.5 ml of methionide/Draekol. Two injections of Omg 7-methyl-8-oxoguanosine (immunostimulant) were administered at 1 Administered at 4 week intervals.

Neutralizing antibody-inducing peptide from the gp7o region of the FLY lumenvelop protein , called 185B, was given to two cats. At 28 weeks after the last injection, 6 vaccines The cat and 6 untreated cats were immunized with glucocorticoids. Suppressing and Live Rickard of Feline Leukemia Virus (R4ckard) We fought against it with bacterial strains. Signs of infection are detected using the main viral core antigen (p) as a capture agent. 27) Monoclonal antibodies and perogysidase-conjugated immunoglobulins against G (LeukAssay)”, New Jersey, Pit mam=Mo Virus protection was determined by ELISA using Monitored before and after anti-dialysis. Five weeks after challenge, immunization with both 1858:KLH was performed. The cats were designated as antigenic (p27 is serum (detected inside). ISP: 1 of 4 cats vaccinated with KLH The patient showed signs of virus in the serum. In the control group (no vaccination) Four of the six cats became antigenetic after challenge. In this way, the application Humans have shown that immunization using ISP conjugates protects cats against FLY infection. I did that first. Same @1=, subject with appropriate viral 15E peptide conjugate Immunization of individuals (Table 3) will protect subjects from subsequent viral infection. .

Table 7 of feline leukemia virus ISP as measured by ConA-induced T-induced field cell proliferation. Sequence of inhibitory active peptide of overlapping peptide conjugate Inhibitory peptide Activation code KSLTSLSEVVLQN・・・・・・・・・・・・・・・・・・・・・・・・・・・ ・・・・・・・・・・・・10 c93KSLTSLSEVVLQNRRC,... ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・++ c94 (C) SLSEVVLQNRI? GLDI・・・・・・・・・・・・・・・・・・ ・・・・・・・・・・・・-/+c95(C)EVVLQNRRGLDILF L･･･････････････＋／＋c96(c )t, oNRRct, p+r-ptoEc−−−・−−−−−−−−−−−・ −−−−−−−/+ LDILFLQEGGLC for c97RR・・・・・・・・・ ・・・・・・・・・・・・＋c98LDILFLOEGGLCAALK--- −−−−−−−−−−−−−/+c99LFLQEGGLCAAL5... ・・・・・・・・・・・・-GLCAAIJEEC to cloooE--- ・・−−−−−−++　clolGLCAALKEECCFLK−bone・・・−− ---c102AALKEECCFLKEEC・・---+++ c103L QNRRGLDILFLQEGGLC・・・・・・・・・・・・・・・・・・ ・10++ (ISP) (-) - No activity, (-/+) - Slight activity, (+) - Considerable activity, (10+) - Excellent I: Activity, (+ 10+) - Very good activity ; (C)-cysteine is not present in the predicted amino acid sequence, but other studies was added for cross-linking.

Tora jl [PI9-HIV peptide with immunosuppressive activity H = derived from the IV sequence Peptide sequences were synthesized using standard in-phase techniques and isolated into mouse Tm follicle mitochondria. (Table 8). Applicant has suppressed activity The peptides observed to have think.

Table 8 Immunosuppression mediated by HIV peptides C232QLTVWGIKQLQA RIL +C233GrKOLQARILAVEI? Y4B QLQARILA VERYL SUPI　LQARILAVERYLKDQQLSUPI-KLH++ C234QARILAVERYLKDQOLISI RILAVERYLKDO QLLGIWGCS-C235AVERYLKDOQLL to IIE34. l AVERYLKDOOLLGIWGC5GilC-E34.1-KLH+++ + E34.2 LKDQOLLGIWGCSGKLIC-5LlP2 QREKR AVGIGALFLGFLG -++5LIP2-F, LH+++ C92TKAKRRWQREKRA C221AVEVGIGALFLGFLGAAFLY peptide: ISP LQNRR(, LDILFLQEG(, LCISP-KLH+++ KLH-glutaraldehyde control Zero KLH - Peptide linked to KLH via glutaraldehyde.

(-) - absent: (+)・weak; (+10)・moderate; (10+1)=strong: (÷ ++ Customers are very strong.

The assay procedure performed was performed on murine (C5) cells utilizing the procedure described in Example 10. 8B+/6 line) proliferation of Concanaba-induced A-induced T cells of tile cells.

Example 1 - Assay for immunosuppression - murine (C58BL/6 strain) tile Proliferation of concanavalin A-induced T cells in cells, cell suspension of C57BL/6 strain Prepared by aseptic technique from mice.

2. The tile cell suspension was pre-washed and pre-warmed (37°C) nylon-wool. Applied onto a column (approximately 10' cells 10.6 g nylon-wool). 45 cells Incubate for minutes and elute dropwise with warmed medium. Concentrated T-thin Gathered a group of cells.

3. Collection I; cells (10'/well) were placed in a sterile 96-well microtiter plate. These wells received various concentrations of peptide (400-25 μg/ m+) was added in advance. The plates were then incubated with 5% Co for 300 min at 37°C. I incubated.

4. Next, plate concanavalin A (ConA) mitogen (1/Jg/well). Add to each well at a rate. Control wells are C.

Cells with and without nA were included. The final volume in each well is 20 0 μl. The following illustrates a typical protocol culture constructed: Tile cell = 50. ul/well (2X10@/m+) Peptide dilution: l O Op1/well mitogen (ConA) 50/71/well-(4μg/ml ) 5, then incubate the plate for 3 days at 37°C in 5% CO□ tion.I;. Tritiated (3H) thymidine (1 microcurie/U) wells) was added to each of the wells 16 hours before harvest.

6. The :H thymidine-pulsed plates were then harvested with a PHD cell harvester. Ta. Place the harvester's filter disc into the scintillation vial here. , add scintillation fluid, and replace the vial with radioisotope present. were counted.

7. Proliferation is expressed as counts 7 minutes (CPM) or as a percentage of control proliferation (i.e. It was determined as the CPM/'ConA control CPMX100) of the experiment.

Medium: RPMI 1640 medium L-glutamine (2 mmol) Sodium pyruvate (1 mmol) Centamicin tL acid solution (20 μs/m+) selected heat-inactivated fetal calf blood Kiyoshi (5%) Sato l glossy mouse g22: The basic proliferation assay is performed in 96-well microtiter plates. and murine tile cells (IXIO' cells/well) were treated with mitogens, e.g. Lipopolysaccharide for T cells and for B cells with Concanavalin A (ConA) This involves inducing and activating lymphoid cells, which can then be placed in culture. and multiply it. Lymphoid cell cultures are incubated for 3-7 days depending on the experiment. The nucleotides were incubated for 12-24 hours and the culture was irradiated for the last 12-24 hours. pulsed with deoxyliponucleic acid (DNA) of proliferating cells. ) is incorporated into the Multi-channel cell harvesting equipment harvests individual culture wells. harvested into glass fiber disks at a stand-alone position, and individual disks were coated with a phosphor-containing thin film. in a tilation fluid and detect the presence of radioactivity in a scintillation spectrophotometer. We analyzed the current situation. the level of cell activation, i.e. proliferation, versus non-stimulating in mitogen-stimulated cultures, with respect to that of stimulatory cultures, i.e., cultures of cells alone. It was measured as the amount of DNA-incorporated 1H-thymidine detected. The data is Sterility index (S, 1.) (calculated as experimental count 7 min (CPM)/control culture CPM ) or as the percentage of control proliferation, i.e. experimental CPM/positive Recorded as control (mitogen in cells) x100.

designed to determine the immunosuppressive activity of the peptide; in the assay, the culture were prepared as described above, but at varying concentrations, the inhibitory peptide was mixed into the initial culture. Inhibition of cell proliferation was demonstrated by exposure to 5.1° or peptide. Percentage of control growth in cultures containing inhibitory peptide compared to cultures without (%). Suppression was considered significant when greater than 40%.

These experiments were not restricted to any particular mouse strain;

816B: ISP-mediated suppression: ConA Ba1b/c tile cells (4*g/m+) 816B tritiated with: stimulated in the presence of ISP and the IH-thymidine pair The level of incorporation was determined on the fourth day. 816B=ISP is 12.5 μg/ml The concentration induced significant suppression (60%) of T cells (Figure 1). What is of interest , higher concentrations of peptide, e.g. 100 μg/ml, did not cause inhibition. . These results were reproducible in additional experiments. Test the culture of this Cell numbers and viability are then determined in control and peptide-treated control wells. They were comparable in both respects. This indicates that suppression is caused by It was not a result of putide toxicity.

Further experiments were performed to test the 816B, ISP, and δ16B:ISP peptides. The relative ability of T-cells to suppress T cells was examined. stimulate the cells with mitogens, and Cultured for 4 days in the presence of each peptide. 816B:ISP peptide is Significant inhibition compared to either δ16B (35%) or l5P (28%) , that is, 78% was induced (Fig. 2). ISP peptide conjugated to KLH 816B:ISP only occasionally induces inhibition, whereas 816B:ISP acts as a free peptide. It was active.

Furthermore, the I5P-KLH conjugate at a concentration of 100-200 μg/ml , constantly induced inhibition (data not shown).

In related studies, extension of ISP in the sense of natural viral sequences as well as A peptide in analog form was synthesized. These peptides are longer peptides. Whether the compound is repressive without the need for conjugation to a carrier or whether the repressive activity is Tested to determine if improvements were made. These extended peptide sequences are , LQNRRGLDILFLQEG(:, LCAALKECCFYADH or It was LQNRRGLDILFLQEGGLCAALKECCFLKEE.

B A L B/c Mouse tile cells were incubated in the presence of mitogen (Con A) for 4 days. The cells were cultured in the presence of peptides and with varying concentrations of the two extension peptides. Which pepti Do too! and t: showed no suppressive activity in mouse T cell proliferation assays.

Tributes were stimulated with ConA or lipopolysaccharide in the presence of δ16B:ISP. culture Materials were assayed on days 4 and 5. 816B: ISP peptide is T cell (Figure 3A), but not B cell proliferation (Figure 3B) .

In additional experiments, gG secretion by JY cells, a human B lymphoblastoid cell line, 816B: Inspect the ISP action. Does not process secreted IgG levels δ16B:ISP suppressed antibody production compared between It was shown that there was no control.

Time kinetics of in vitro inhibition = 816B: During the method of testing ISP-induced inhibition , Applicants have demonstrated that in a series of experiments using murine and human cells, the peptide observed that it did not proliferate. The assay is carried out for 3 days rather than 4 or 5 days. and mitogen-induced proliferation is generally significant after 5 days of culture. It was recognized that this is the case.

Therefore, experiments demonstrated that biological effects on the ability of 816B:ISP to suppress T cell proliferation Experiments were performed to determine the effect of incubation time. As the results dramatically show , suppression was not observed in 3H cultures and occurred only after 4 days (Fig. 4, 9°). This shows that peptides interact in metabolic pathways and exert this effect. It takes a certain amount of time to complete.

In addition, the proliferation of pokeweed mitogen (PWM)-stimulated human tile cells was 816. B: Found to be suppressed by ISP.

Recognition of δ16B:rsp by anti-peptide serum: Applicants have obtained anti-16B serum (R Affinity-purified rabbit antiserum against 112-5)8 and ISP peptide (AP5 5g-28), as well as a monoclonal antibody (9-14F2 -3) δ16B: The ability to bind to ISP was tested and each antibody binding site remained intact after combining the 816B and ISP sequences. . Maf: Control affinity purified rabbit antibody against the c-abl oncogene (A P67) was used. As the results obtained show, both anti-16B and anti-I The SP antibody reacts with δ16B=ISP (Figure 5).

'$3 experiment Protein sequence alignment method: also includes sequences related to selected polypeptides. The identification of these proteins was carried out using the PC gene (Gene Released from Intelligeneties, Inc. (re　ease)4゜05〕 SCANSIM program (modification 3.02 ) and the Swiss Protein Database (Release 2). choice Due to the severe alignment, Nedleman-Wunsch Application of man-Winsch algorithm (PEP/ALIGN) Bionet (from Intelligenetics, Inc.) Bionet).

Polypeptide sequence: The polypeptide is the predicted sequence of the FLY envelope gene precursor. Protein sequences E Elder et al., Virol J. ogy), 46:871-880.1983] and conventional solid phase methods. It was synthesized by

Generation of antisera against synthetic polypeptides Keyhole lymphase generation of two synthetic polypeptides keyhole limpet hemocyanin (KLH) and l=1 in 0.04% glutaraldehyde for 30 min at room temperature. Combined by mixing between. New sealant for your own rabbit, complete froin Inject 200 pg of dialysed conjugate in adjuvant, then inject for 70 days. Two or more booster injections of 200 μg in adjuvant were administered.

[Labeling of proteins by metabolic incorporation of 3ssl amino acids: immunoprecipitates %2X10' cells for each of L-lysine, L-leucine, L-glutamic acid 2.0 ml of methionine supplemented with 5% dialyzed calf serum albumin. 0.5 mC1 of [s15]methionine (or Metabolically labeled with ["Slmet/["Slcys mixture]].

■Detection of Smet-labeled antigen by immunoprecipitation: 2X10'll [1. O m+ lysis buffer (0.05% SDS, 1% Triton X-100, 1% DE Oxycholate, 1 mmol EDTA, 150 mmol NaCl, 100U K tracinol/ml, 50 mmol MOPS% pH 7.0) dissolved in and 10 for 10 minutes. Cleaned by centrifugation at OOOrpm (Sor vall 5S34 rotor).

Antigen was precipitated by addition of 0-020 ml of antiserum for 18 hours on ice. immunity The complex was added to 0.100 ml of Sepharose Nibrotiin A. [Pharmacia] with Rose (Sepharose) ) - Wash the immunized complexes with cell lysis buffer and add 0-050 ml of SDS sample buffer. solution (2% SDS, 10% glycerol, 0.125 mol Tris-MCI, denatured by the addition of pH 6,8) and immersed in rapidly boiling water for 3 minutes. Ta. The beads were pelleted by centrifugation and the supernatant was diluted 20x in cell lysis buffer. and the immunoprecipitation step was repeated. The resulting immune complexes were washed with 10% 2-mer The cells were assembled with 2% SDS sample buffer containing captoethanol. The sample is Stacked gels of 3% and single concentration (12%); gradient (IO-20%) separation gel "Laemmi, Nature, 227- 5DS-polyacrylamide game using 1680-685 (1970)] The cells were separated by electrophoresis. Radiolabeled antigen to conventional fluorographic methods Therefore, it was detected.

Immunocytochemical detection of antigens Rabbit antibodies against two synthetic peptides were purified in two steps. Ta. The immunoglobulin fraction was extracted from serum with 25% to 50% (w/v) ammonium sulfate. Collected from the precipitate. After dialysis against phosphate buffered saline, the sample was epoxy activated. Peptide affinity color prepared with 48 [Pharmacia] applied to the system. Wash the column with phosphate buffer/0.5M KCI and Antibodies were eluted with 1 molar acetic acid containing 3 molar phosphate buffer, pH 8. life The sex fractions were pooled, dialyzed, and concentrated by ultrafiltration. immunocytochemical The procedure includes a fully labeled second antibody and The silver enhancement described in the issue was used.

Identification of proteins containing sequences homologous to ISP: I of FLVp15E The SP domain is highly conserved among RNA tumor viruses. whole swiss star SCANSIM program search (release 2) of protein database is FLY - Envelopes of nine viruses with peptide domains very similar to ISP A loop sequence was identified. at the carboxyl terminus of all nine env peptides. The consensus sequence rQNRRGLDXLJ is used to analyze the protein for related sequences. We searched the quality database. Table 9 below shows the keywords “interferon”, Swiss protein selected using “interleukin” and “tumor necrosis factor” Search for QNRRGLDXL-containing sequences in a subset of quality databases summarize the results. Similar results (not shown) were obtained from the Protein Information Resource (PI PEP/5EARCH with an equivalent subset of R: Intelligenetics) /ROM0LOGY.

Table 9 Interferon/interleukin with highly conserved portions of viral IsPs Kin/tumor necrosis factor alignment (QNRRGLDXL) Position Array Description 33-41 RNRRALILL human IFN a-10 precursor 33-41 RN K RA L K V L Mouse IFN a-2 precursor 33-41 RN K RA L T L L Mouse IFNσ-1 precursor 33-41 D N RRT L M L L Human IFNr-1 precursor 33-41 GNRRA LI LL Human IFN a-4 precursor 33-41 G Human IFNα-5 precursor 33-41 GNRRAL I LL human IFN a -5 Precursor 33-41 GNRRAL I LL Human IFN a -9 Precursor Body 40-48 v P scale SLE 5k Human IFNτ induced protein precursor 33- 41 NNRRTLMLM human IFN (+-8 precursor 33-41 G S R RT L M L L Human IFN a-2 precursor 10-18 GSRRTL MLL human IFN a-3 precursor 128-136 VQRKAIHEL human IFNrR precursor 30-38 QRR5dan ALC bovine IFNβ-2 precursor 11 0-118 K K RD D F E K L Human IFNτ precursor 114- 1220QLNDLEVL Human IFN a-4 precursor 126-134 VQ RQAFNEL Mouse IFNτ precursor 105-113 LN scale RANA L Human TNF Precursor 72-80 0KTQAISVL Human IFM a-9 Precursor body 72-80 0KTQA I SVL human IFM a-10 precursor 127 (351QHJ AVNE L 5'/) IFMr front IK body 126-134 EERVGETPL Human IFMa-10 Precursor 72-80 0KAQAISV L human IFM a-4 precursor 72-80 0KAQA I SVL human I F) Ja-5 Precursor 72-80 Stand KAQAISVL Human IFM a- 6 Precursor 16-24 0KAQA I SVL Human IFM a-7 Precursor 7 2-80 0KAQA I SVL Hime F) J a-8 Precursor 42-50 VQMRRLSPL Mau7. IFMa-1 pre-N body 77-85 PE5KAIK NL Human IFMτ-induced protein precursor l1l-119D L HQ Q L N D L Mouse] FMa-1 Precursor 42-50 AQMRRLP" 4 Mouse IFM a-2 precursor 111-119 DLHQQLNDL Mouse I Application of a stricter alignment program than the FM a-2 precursor (Nedrech Mu/Winch [PEP/5EARCH/ALIGNI is the virus p15E I Amino-terminal amino 8 of the SP domain and human alpha interferon sequence 21-47. Site-directed mutagenesis of oligonucleotides Recent studies using different molecules have shown that the receptor binding site for human alpha interferon is Indicates that it exists within the amino terminal l O-44 amino acids; Shaff e rmn et al. Chemistry (J, Biol, Chem,), 262.6227-6273 ( 1987)], Applicants believe that biologically active ISP and ISP-related antigens are Those that act by cell receptors for rufa interferon Think of it as an intermediary.

ISP-associated antigens are released in the growth medium of various human leukemia cell lines: four white Hemophilia-inducing cell lines were metabolically labeled with [”S]met for 4-6 hours and allowed to mature. 40 hours in total growth medium [Chase] Lf::Cell line! ! HL60 Human acute myelinogenic leukemia Raji Human Purkit 8 Burki tt) Lymphoma 562 h) Rh''' chronic brain myelin developing leukemia erythroid leukocyte blasts Onset F　74　F　LV''J)　jJ　Tumor-conditioned growth medium The soil was cleaned by low and high centrifugation. Then the supernatant from which the antigen can be obtained Immunoprecipitated from fractions of t; Purified antigen was run on a 10-20% gradient SDS gel. separated and detected by fluorography. antigen in soluble 110, Conditions for all leukemia cell lines tested, including FLY'FL741i1 cells. detected in the cultured growth medium.

Duplicate FLV I SPI; tRa j: +7 from m cell) 110.000 da The effect of the root ISF peptide is on [”S]me-labeled FLV gPr’″ ″/p15E and Raji cell antigen blocking FLV ISP antiserum We compared the ability to As the results summarized in Table 1O show, overlapping FL Y peptide blocks detection of feline virus and human leukemia cell induction. They show very similar abilities.

Table 1O Immunoprecipitated FLV-p15E and Raji ll0K protein FLV Blocking of FLY peptide in recognition of ISP antiserum [Detection of Slmet antigen] Out Peptide sequence FLV p15E Raji No ll0K ++4+ ++ FLV ISP LQNRRGLD ILFLQ FGGLCF198 VVL QNRRGLD IL ++ +F]97 VLQNRRGLDILF +F1 95 0NRRGLD ILFLQF194 NRRGLD ILFLQ EF 186 LD ILFLQ EGGLC++++ ++F’187 D ILF LOEGC, LCA ++++ ++F188 1LFLQ EGGLCAA + -F189 LFLQ EG (, LCAAL +++++ ++F190 F LOEGGLCAALK +++++ 10 + (-) - not present; (+) = weak; ( ++)=moderate; (+++++)=very strong.

T cell mitogens (PHA) are found in normal human PBL [3″S1met-labeled 110 Stimulates the synthesis of antigens: Histobark from normal healthy donors paque) enriched peripheral blood leukocytes with 10 μg/ml phytohemagglutinin. It stimulated me. After 4 days of culture, unstimulated and stimulated cells were reduced to 4% [" Labeled with Slmet and lysed for immunoprecipitation. What the results show According to and very high levels of antigen were detected in mitogen-stimulated cells. ;.

It was not detected in Albino 3T3 cells. early immunity Precipitation experiments were performed using 4-h [”S]met-labeled extracts to Detect whether the K antigen is detected in any cell line. ll0Kta Protein was clearly detected in A431 cells, but not in 3T3 cells. Not detected.

The present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding. Although described in detail, it is clear that changes and modifications may fall within the scope of the appended claims. can be administered. For example, the peptides of the invention may contain conservative or non-conserved Changes can be made by substituting conservative amino acids. Similarly, from the original The clear peptides can be subjected to various changes, such as amino acid insertions or deletions. where such changes can lead to the desired immunosuppressive activity in vitro or in vivo. It may or may not have a substantial effect.

FIGURE 1 200 100 50 25 12j 6.25 3.1 1.5FIGLIR E2 FIGURE 3A FIGLIRE 3B FIGURE 4 FIGυRε5 international search report °"l#h@MI　＆-ゝ-"12Cτexposure"I　u　:τ6

Claims (1)

[Claims] 1. consisting of a peptide having less than about 40 amino acid residues; The group consists of an amino acid sequence of at least five amino acid residues, said amino acid sequence is the range of the amino acid sequence LQNRRGLDILFLQEGGLCAALKEECCF. An immunosuppressive peptide selected from the amino acid sequences contained within. 2. An immunosuppressive compound comprising a peptide according to claim 1 conjugated to a carrier molecule. . 3. consisting of a peptide having less than about 40 amino acid residues; The group consists of an amino acid sequence of at least five amino acid residues, said amino acid sequence teeth, [There is an array] Amino acids contained within the range of an amino acid sequence selected from the group of amino acid sequences consisting of an immunosuppressive compound selected from amino acid sequences, said peptide being conjugated to a carrier molecule. thing. 4. The carrier molecule is keyhole limbet hemocyanin (keyhole Iim). 4. The compound according to claim 2 or 3, which is a pet hemocyanin. 5. Claims 2 and 3, wherein the carrier molecule is conjugated to the peptide with glucuraldehyde. or the compound described in item 3. 6. consisting of a peptide having less than about 40 amino acid residues; The group consists of an amino acid sequence of at least five amino acid residues, said amino acid sequence is the amino acid sequence QREKRAVGIGALFLGFLG or QLTVWGIK An immunosuppressive peptide selected from the amino acid sequences contained within the scope of QLQARI Do. 7. Antioxidants of peptides having the amino acid sequence LQNRRGLDlLFLQEGGLC An immunosuppressive peptide consisting of antigenic determinants homologous to the original determinant. 8. a first amino acid sequence of at least five amino acid residues, said first amino acid sequence is from the amino acid sequence contained within the range of the amino acid sequence AKLRERLKQRQQ selected, and at least one other amino acid of the at least five amino acid residues. amino acid sequence, other amino acid sequences mentioned above, [there is a sequence] Amino acids contained within the range of an amino acid sequence selected from the group of amino acid sequences consisting of an immunosuppressive peptide consisting of selected from the amino acid sequences. 9. Of the amino acid sequences contained within the range of the amino acid sequence AKLRERLKQRQQ The amino terminal is [There is an array] Amino acids contained within the range of an amino acid sequence selected from the group of amino acid sequences consisting of The immunosuppressive peptide according to claim 8, which is linked to the carboxy terminus of the amino acid sequence. Chido. 10. Amino acid sequence included within the amino acid sequence AKLRERLKQRQQ The carboxy terminus of [There is an array] Amino acids contained within the range of an amino acid sequence selected from the group of amino acid sequences consisting of The immunosuppressive peptide according to claim 8, which is linked to the amino terminal of the amino acid sequence. . 11. Amino acid sequence included within the amino acid sequence AKLRERLKQRQQ Each of the carboxy-terminus and amino-terminus of [has the sequence] Amino acids contained within the range of an amino acid sequence selected from the group of amino acid sequences consisting of 9. The immunosuppressive peptide according to claim 8, which is linked to a amino acid sequence. 12. amino carboxy of the amino acid sequence that falls within the acid sequence AKLRERLKQRQQ The amino acid sequence connected to the terminal is the amino acid sequence AKLRERLKQRQQ. is the same as the amino acid sequence bound to the amino terminus of the amino acid sequences contained within the range. The immunosuppressive peptide according to claim 11. 13. Amino acid sequence included within the amino acid sequence AKLRERLKQRQQ The amino acid sequence attached to the carboxy terminus of Amino acid sequence that binds to the amino terminus of the amino acid sequence contained within the range of KQRQQ The immunosuppressive peptide according to claim 11, which differs from the column. 14. The immunosuppressive peptide according to claim 8, which is cyclic. 15. A polymer comprising repeating units of the peptide according to claim 8. 16. A shimmer comprising a repeating unit of the peptide according to claim 8. 17. Amino acid sequence AKLRERLKQRQQLQNRRGLDILFQEGG Immunosuppressive peptide consisting of LC. 18. Amino acid sequence AKLRELKQRQQLQNRRGLDlLFQEGGL An immunosuppressive peptide consisting of C. 19. A peptide having the amino acid sequence LQNRRGLDILFLQEGGLC The antigenic determinants of antibodies raised against the polypeptide have affinity for A purified polypeptide expressed in animal cancer cells. 20. Claim 19 having an apparent molecular weight of about 110,000 Daltons. Purified polypeptides. 21. Claim 19 having an apparent molecular weight of about 35,1000 Daltons. Purified polypeptide. 22. A purified polypeptide encoding the purified polypeptide of claim 19. Nucleic acid sequence. 23. A purified polypeptide encoding the purified polypeptide of claim 20. Nucleic acid sequence. 24. A purified polypeptide encoding the purified polypeptide of claim 21. Nucleic acid sequence. 25. Affinity for the polypeptide according to claim 19, 20 or 21 Antibodies with. 26. The polyclonal antibody according to claim 25. 27. The monoclonal antibody according to claim 25. 28. has an affinity for the polypeptide according to claim 19, and has an affinity for the polypeptide according to claim 19, and Purified protein present on the surface of production cells. 29. consisting of the antibody according to claim 25 and a pharmaceutically acceptable carrier. Therapeutic composition. 30. Effective immunosuppressive effect of the therapeutic composition according to claim 29 on a subject immunologically or hematopoietically suppressed subjects How to treat. 31. The purified polypeptide according to claim 19, 20 or 21, or a peptide having an amino acid sequence of at least 5 amino acid residues; the amino acid sequence is included within the purified polypeptide and is pharmaceutically acceptable. A therapeutic composition comprising a watery carrier. 32. Administering to a subject an effective immunosuppressive amount of the therapeutic composition according to claim 31. A method of suppressing a subject's immune system, comprising: 33. The purified protein according to claim 28, which has immunosuppressive peptide binding activity. A therapeutic composition comprising a portion of a protein and a pharmaceutically acceptable carrier. 34. Effective immunosuppressive blocking of the therapeutic composition according to claim 33 on a subject for immunologically or hematopoietically suppressed subjects, which consists of administering How to treat. 35. A peptide having the amino acid sequence LQNRRGLDlLFLQEGGLC has an antigenic determinant that reacts with an antibody raised to comprising detecting cells from a sample expressing a polypeptide; A method for detecting cancer cells in a sample, in which tido is expressed in mammalian cells. 36. A peptide having the amino acid sequence LQNRRGLDlLFLQEGGLC a polypeptide having an antigenic determinant that reacts with an antibody raised against said polypeptide; The peptide is expressed in a mammalian cell, or the peptide contains said antigenic determinant. The repeptide part is detected in the body fluid collected from the subject. A method for diagnosing cancer in a subject. 37. Peptides and pharmaceuticals according to claims 1, 6, 7, 8, 17 or 18 A vaccine consisting of a biologically acceptable carrier. 38. Administering an effective immunizing amount of the vaccine described in claim 37 to a subject. A method of immunizing a subject against a retrovirus. 39. A compound according to claim 2 or 3 and a pharmaceutically acceptable carrier. A vaccine consisting of 40. Administering an effective immunizing amount of the vaccine according to claim 39 to the subject. A method of immunizing a subject against a retrovirus. 41. Peptides and pharmaceuticals according to claims 1, 6, 7, 8, 17 or 18 A therapeutic composition comprising a pharmaceutically acceptable carrier. 42. Administering to a subject an effective immunosuppressive amount of the therapeutic composition according to claim 41. A method of suppressing a subject's immune system, comprising: 43. The bebutide according to claim 2 or 3 and a pharmaceutically acceptable carrier. A therapeutic composition consisting of the body. 44. Administering to a subject an effective immunosuppressive amount of the therapeutic composition according to claim 43. A method of suppressing a subject's immune system, comprising: