JPH01500594A - Peptide fragments of organ-specific neoantigens - 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] Field of invention of peptide fragments of organ-specific neoantigens The present invention relates generally to the diagnosis of cancer in humans, and more particularly to the diagnosis of cancer in humans. can mediate leukocyte adhesion inhibition in the serum of patients affected by cancer The present invention relates to the preparation of antigenic peptides that are fragments of sexually transmitted tumor antigens.

With the emergence of new and effective methods for cancer treatment, it is important to provide early diagnosis and It is becoming increasingly important to be able to monitor the status of cancer during treatment. It has become important.

To be effective, many regimens must be started early in the illness. The extent of the tumor should be monitored in order to adjust the regimen accordingly during the treatment period. It is important to monitor

One approach to diagnosing and monitoring cancer is the use of biological specimens, more specifically blood samples. Based on the measurement of tumor antigens in serum and tissue samples. Such tumor antigens, called CARs, are produced based on tumor cells and their characteristics. substances such as proteins, glycoproteins, and polysaccharides. The tumor antigen is , cellular metabolites, cell surface antigens, etc., which typically enter the circulation under a variety of circumstances. It can be a cell surface antigen that can enter the cell. Measurement of tumor antigens secreted into serum in this way may be a diagnosis of malignancy.

To date, a substantial number of tumor antigens have been identified, but no single tumor antigen is associated with cancer. To date, no completely reliable criteria for diagnosis and monitoring of cancer have been provided. So Therefore, it can be used alone or in combination with other tumor markers for cancer diagnosis and detection. It is desirable to identify additional tumor markers that can be used.

organ-specific neoantigen en) A specific 40kD antigen called p40 (OSN p40) ・Dr. David Thomson's research group (Montre alGeneral　Ho5pital,　Montreal+　Quebec , Canada). OSN p4o is on the membrane of solid tumor cells and peripheral vascular leukocytes of patients suffering from these types of tumors ( Trigger of immune-mediated leukocyte aggregation inhibition (LAI) in PBL). OSN Even though p40 is promising as a tumor marker, its purification is difficult and As such, its use in leukocyte aggregation inhibition assays is limited. Because one type Antigens from tumors, such as lung tumors, can be used to treat more than one type of cancer, such as lung cancer and bladder cancer. It may also induce LAI in the serum of patients suffering from cancer, lung cancer, and breast cancer. It is.

Therefore, it is possible to specifically identify OSN p40 from various types of tumors in patient samples. It would be desirable to provide improved reagents that include antigens and antibodies that can be detected visually. .

Among other things, specificity for certain tumor types and cross-reactivity with other tumor types It would be desirable to provide such reagents that do not have any properties.

Description of conventional technology Extracts from cancer cells can be used to stimulate peripheral blood white blood cells from patients affected by certain forms of cancer. (Halli-day and Miller' (197 2) Int, J. Caneer 9: 477-483), such extraction Tumor antigens derived from substances are called organ-specific neoantigens (Grosse r and Thomson (1975) Cancer Res, 35: 257 1-2579). Organ-specific neoantigens have been shown to be membrane proteins and is not solubilized by crude extraction techniques (Grosser and Thomson (1979) ibid.] can be solubilized by limited papain digestion (Th omson et al. (1976) Cancer Res.

36: 3518-3525). At least some organ-specific neoan Thigen is shed from the cancer cell surface and can be detected in both leukocytes and urine (Gr Osser and Thomson (1976) Int, J, Cancer 18 : 5B-66). Certain organ-specific neoantigens are derived from both white blood cells and urine. isolated (Lopez and Thomson (1977) Int, J. Caneer 20 2 834-848; Thomson et al. (1980) Br, J, Cancer41: 86-99: and Thoa+son et al. 1980) Europe, J. Cancer 16:539-551). organization Cultured cancer cells express organ-specific neoantigens and spend the antigens released into the medium (spent n+edi, um [Lajzerowiez et al. 1982) J, Urol, X28: 1122-1129: and Khosra vi et al. (1983) Transplantatiotv 35: 258-2 66).

Inhibition of leukocyte aggregation (LAN) in peripheral blood leukocytes (PB[,) of patients with lung cancer] Purification of an approximately 40 kD antigen from lung cancer cells that is responsive to Thomson's on) et al. (Intj.

Cancer 35: 707-714 (1985)), colorectal disease patients A similar antigen that can induce LAI in PBL has been purified from colon cancer cells. (Artigas et al. (1986) Cancer Res, 46: 18 74-18813.

U.S. Patent No. 4,426.446 features maximization of cAMP levels in white blood cells A method for performing a leukocyte aggregation inhibition assay is described. Again Thomson et al. (J, Natl, CancerInst, 75:995-1 003 (1985)) and Laba Teya and Thomson ( See also (Ibid. 75: 987-994).

Summary of the invention The present invention provides methods and compositions useful for diagnosing and monitoring various cancers. . The composition comprises peptide fragments having up to 100 amino acids, generally up to 50 amino acids. of amino acids, and the amino acids are effective in the tissues of specific body organs. Leukocyte aggregation inhibition (LAN) in peripheral blood leukocytes (PBL) of patients with cancer Organ-specific neoantigens (O 3N's). The OS derived from a different body organ Although N's are structurally similar and share common skeletal determinants, they one or more unique antigens that can serve as a standard for identifying the cellular origin of Characterized by determinants. The peptide fragments of the invention are generally specific to cellular origin. Contains characteristic unique antigenic determinants that are characteristic of more than 1.2 tissue types However, in some cases, the cause may be The above common decision allows detection of O5N regardless of the cellular origin of the tumor. It would be desirable to provide peptides comprising such groups. Patients affected by lung or colon cancer Typical peptides that can clearly induce LAT in PBL from It is written in the column. Peptides that exhibit immunological cross-reactivity between one or more types of OSNs Tides were also prepared.

Diagnosis and monitoring of cancer is based on OS in biological samples, typically serum, urine, etc. This is performed by detecting N's.

Antibodies against the peptides are supplied and tested using various immunological techniques, e.g. Riza (El, ISA: enzyme-linked immunosorbent assay), radioimmuno Assays and enzyme immunoassays are used to detect the above O3N. Can be used. Utilizes immunological reagents that are specific for organ-specific antigenic determinants. By this, the cellular origin of the cancer can be determined. When the cellular origin of cancer is elucidated have also used immunological reagents specific for common antigenic determinants of cancer O5N. There will be. Alternatively, the peptides of the invention may be used to detect the presence of cancer in a patient and/or Used in leukocyte agglutination inhibition assays to detect and diagnose cellular origin obtain.

Brief description of the drawing Figures 1 and 2 show the production of carboxymethylated lung p400SN by limited acid hydrolysis. This is a reversed-phase high performance liquid chromatography (HPLC) map of different peptides. .

Figure 3 shows the result of cyanogen bromide decomposition of carboxymethylated lung p4QO3N. This is a reverse phase liver LC map of peptides.

Figure 4 shows the results of tolbutophan degradation of carboxymethylated lung p400SH. This is a reversed-phase HPLC map of peptides.

Figure 5 shows arginine in acylated and carboxymethylated lung p4005N. This is a reversed-phase HPLC map of peptides generated by trypsin digestion.

Figure 6 shows the limited chymolybcin degradation of carboxymethylated lung p400SN. This is a reverse-phase HPLC map of the peptides produced.

Figure 7 shows white blood cells from patients with lung cancer, malignant melanoma, or benign disease. Dose response curve for p400SN.

Figure 8 shows the results of cyanogen bromide degradation of carboxymethylated colonic p400SN. This is a reverse phase liver LC map of peptides.

Figure 9 shows the results of tryptic digestion of carboxymethylated colonic p400SN. This is a reverse phase HPLC map of peptides.

Figure 0 shows the trypsin degradation of reduced and carboxymethylated lung p400SN. This is a reverse phase HPLC map of peptides produced by

Description of preferred embodiments The present invention provides an organ-specific neoantibody that can inhibit leukocyte aggregation as described below. Antigenic peptide fragments of O3N's are used for the diagnosis and monitoring of human cancer. It is something that can be used in a variety of ways.

The O5N's are in the range of about 35-45 kd, typically in the range of about 37-43 kd. , more commonly has a molecular weight of about 40 kd, and is found in the lungs, colon, breasts, and bladder. derived from malignant cells or fetal cells from various human body organs, including The above difference The O5N″S from the organs are structurally and immunologically related.Structurally related Regarding gender, the amino acid sequences of OSN's of various nuclear species are at least 50% , generally at least 60% homology, and often more than 75% homology. It means to show. In terms of immunological relevance, the above from different organs OSN's share at least one common epitope site and generally have multiple share an epitope site, but typically much less than all antigenicity-determining sites occupy the position. However, in the case of OSN’s from certain organs, e.g. lungs or breasts There are 0 molecules that can be virtually identical both structurally and immunologically. The approximately 40 kd OSN's of the invention may be described below as p40.

Leukocyte aggregation inhibition c+:m is a physiological response mediated by various chemical affinities. be. LAI also stands for in vitro inhibition of leukocyte aggregation; cell-mediated immunity as disclosed in U.S. Pat. No. 4,426,446. It can be used as a standard for further analysis. This disclosure is incorporated herein by reference. Incorporate. In antigen-induced LANs, the response is induced by sensitized leukocytes or immune Depends on antigen recognition by white blood cells. When binding to the antigen, leukocytes use mediating factors ( med i a tor) and the mediator is otherwise aggregating. Approximately 30% of bystander cells will have Prevents agglomeration. Antigens recognized by white blood cells from human tumors (OSNs) It is organ specific. That is, OSNs derived from malignant cells from individual body organs are The organ may induce LAI in white blood cells from patients affected by cancer. However, it is generally not induced in leukocytes from patients with cancers of other organs. However, some redundancy is observed. For example, from cancerous lung cells. OSN from patients with breast cancer as well as in PBL from patients with lung cancer. It will also partially induce LAI in PBL.

The peptide fragments of the invention are both haptenic and antigenic, and are naturally occurring Contains 6-100 amino acids found in 5N, typically 9-75 Contains amino acids, more commonly 12-75 amino acids. the peptide The fragment contains one or more epitopes corresponding to unique antigenic determinants characteristic of the above OSN. These epitope sites may contain or antigen specificity for the epitope site, or both. will be recognized. In general, epitope sites are antigenic determinants characteristic of the cellular origin of OSNs. fixed base (to allow determination of the cellular origin of solid tumors). deer antigenic determinants common to two or more O3N's, sometimes of heterogeneous cellular origin. It may also be preferable to provide peptides having an epitope site corresponding to .

The peptides may be natural, i.e. fragments of OSN isolated from natural sources. Natural peptides may be derived from natural sources, e.g. those producing OSN, or may be synthetic. From cell lines known to be It can be isolated by photography. Preferably, obtained according to the present invention Polyclonal or monoclonal antibodies can be prepared with appropriate affinity using well-known techniques. For example, such a technique can be used to prepare a tea column. Practical Immunology (Pr. Iudson) and Bay (Pr. actical publica-tion, Oxford, United Kingdom, 19 80. Chapter 8). To isolate the peptides The specific method is shown in the Examples section below, and the peptides are The as-isolated protein is then chemically or enzymatically treated as described in the It can also be obtained by cutting into.

However, it is difficult to isolate intact OSN’s from natural sources; Preferably, synthetic fragments are prepared based on the natural O5N sequence. The natural O5N The column sections are shown below for lung and colon OSH. Natural OSN and immunological Cross-reactive synthetic polypeptides can be produced by either of two general means. It can be prepared. First, about 60 amino acids or less, more commonly about 3 Polypeptides with zero or fewer amino acids contain amino acids in a growing chain bound to a solid phase. can be synthesized by the well-known Merrifield solid-phase synthesis method in which rifield (1963) J, Ai, Chen+, Soc, 85: 21 49-2156).

The second method for synthesizing the peptide of the present invention is to synthesize the desired region of the O5N gene. the O3N gene, with expression in cultured cells of a recombinant DNA molecule encoding the O3N gene. The progeny may be natural per se or derived from cDNA or genomic libraries. obtained using a degenerate probe based on the known amino acid sequence described above from Lee. It may also be a synthetic product using a natural gene.

Suitable cDNA and genomic libraries include N-! Known humans containing the gene It can be obtained from cell lines such as the NCl-H69 lung cancer cell line. different from the above Alternatively, polynucleotides can be synthesized by well-known techniques. For example, short single strand D The NA fragment was generated using the phosphoramidifte method (Beaucage and Carruthers). 1981) Tett.

Letters 22: 1859-1862). Next, two The heavy chain fragments synthesize complementary strands and the strands are annealed together under appropriate conditions. or by using DNA polymerase to generate appropriate primer sequences. obtained by adding complementary strands to the sequence.

Natural or synthetic fragments encoding the desired O3N fragments can be introduced into in vitro cell culture. will be incorporated into the NA construct, generally The DNA construct is suitable for replication in a unicellular host, such as yeast or bacteria. However, it is also possible to introduce and assemble into cultured mammalian or other eukaryotic genomes. may be intended for inclusion. DNA construct prepared for introduction into tR bacteria or yeast the replication system recognized by the host, co-coding the desired polypeptide product. The OSN DNA fragment to be encoded, the translocation linked to the 5'-end of the O5N DNA sequence. transcription and translation initiation control sequences, and transcription and translation initiation regulatory sequences linked to the 3'-end of the O5N sequence. Translational termination control sequences can be included. The transcriptional regulatory sequence is a heterologous sequence recognized by the host. It can contain sexual bromotar. Preferably, the insertion for the above OSN DNA sequence is The entry site contains the replication system and transcription and translation regulatory sequences together. Any available expression vector can be used.

4 corresponding to lung and colon O5N, as described in more detail in the Examples section below. -21 unique peptides with 55 amino acids were prepared from natural sources. child The sequences of these peptides (using standard abbreviations) are as follows: (I) GTFLNFX+ILN GKX3hDTYGPLTGYN GYKXI (II[)　LNKKKGGKEV　NKEV(Vl)　NNEANEFMEA L (■) AAWK (■’) INENAKDN (IX) IVTNPIPY (X I) NTFNNETILHGKDAXQ (XI) NTFLNVTX , LX, G (XIII) QSVYDPTVQA N (XIV) VSQGQ W.D.K. (XV) FYVDGTDSDDP LVK (XVI) YVDGTDSDPL VK(X■) WNGNVT (X■)AS kaiTDEN)IK (XIX) IDNFQK (XX) FTTLNBDR (XX 1) IFVETPY X = unknown amino acid X3-G or LXI-T or G x, N or E xi - N or A X, = Q or H Among these 21 peptides, peptides m, rv, v, and X determine the original protein skeleton. It was found to have strong reactivity with anti-p40 monoclonal antibodies directed against the I was taken out. Peptide ■ is recognized by white blood cells from lung cancer patients, but is not recognized by other cancers. Pulmonary p4o determinants of OSN not recognized by leukocytes from affected patients contains. A synthetic fragment of peptide ■ was prepared as described in the Examples section. , this then stupefies the same activity.

A peptide of 12 amino acids derived from colonic p400SN, as described in the Examples section. A peptide was also isolated (corresponding to peptide x), which has not been sequenced. and synthetic peptides were prepared on the basis of that sequence. Synthetic Colon Pep Chido is the following array: FYVDGTDSDDP LVK It had Using the above synthetic peptide, a very strong positive reaction was detected with the peptide itself. In addition to this, we prepared an antigen that reacts positively with both colon and lung p40.

The peptide fragments generally have at least about 8 0% homology, more commonly at least about 90% homology, and further often have identical sequences. Limited substitution of amino acids is especially This can be done if it does not affect the antigenic properties of the putide. The peptide is They may be modified at one or more sites to enhance their properties. This is more than 1 This includes insertion, deletion, or modification of amino acids or groups. The page 1. Proteins, other peptides, polyamino acids, solid supports or small Other amino acids or can also add groups. The peptide may be modified to adjust its overall hydrophilicity. It is also possible to add a group for For example, hydrophobic amino acids or valmitic acid Fatty acids such as Adding to promote ing m1eells forn+ation) Can be done. The amino terminus may be free or acylated, and The carboxyl terminus may be a free acid or a carboxamide. Ma Additionally, the peptide may contain other amino acid sequences, such as immunogens useful for specific purposes. Can be combined.

To be useful in the detection methods of the invention, the peptides are generally substantially pure. Obtained in form. i.e., is typically about 50% pure/w or higher and interferes with Proteins and contaminating bacteria have been virtually eliminated. Preferably O3N pep at least 80% −/−1 and more preferably at least about 95% G Isolated or synthesized with a purity of 11/-.

Using standard protein purification techniques, a small number (99% -/- homogeneous peptide For example, the protein has immunoadsorption affinity or chromatography. It was purified by using the antigen described below. The affinity black Matography first binds the antigen to a solid support, and then this bound antigen. Original and O5N protein sources, such as naturally produced OSN or recombinant O5N By contacting the lysate of cells producing OSN, which is obtained by introducing a DNA molecule, This will be implemented by

The O5N peptide of the present invention is useful for malignant diseases, especially solid 11IiT&, such as lung cancer, Can be used to detect and monitor intestinal and rectal cancer, breast cancer, prostate cancer, liver cancer, etc. . Detection and monitoring can be accomplished by analyzing appropriate biological specimens. It can be carried out by I assay or known immunological assay. LAN assay can be carried out as described in U.S. Pat. No. 4,426,446, which disclosure is incorporated herein by reference. Incorporated herein by. Immunological techniques detect antibodies in a patient's serum The peptides can be used as antigens in various biological specimens such as We now turn to the preparation of antibodies from the peptides to enable the detection of antigens.

The use of the above peptides in LAN assays is described in detail in the Examples section below. This is similar to using an intact p400SN. The LAN Assay is a mixture of peptides, e.g. a cyanogen bromide decomposition mixture of native p400SN. It can be implemented by

The assay can also be performed with purified peptides, natural or synthetic. Ru.

Antibodies against the P2O0SN peptide can be isolated from a wide variety of vertebrates using conventional methods. It can be obtained by injecting a manufactured peptide. Preferred vertebrates include: Mice, rafts, sheep, and goats are included, and mice are particularly preferred.

Generally, these animals are treated with continuous exsanguination to maintain improved titer and specificity. Blood samples are taken periodically. The antigen can be injected intramuscularly, intraperitoneally, or subcutaneously. I can do it. Generally, excipients such as complete or incomplete Freund's adjuvant is used. Monoclonal antibodies can be prepared if desired.

To obtain monoclonal antibodies, lung cells from immunized vertebrates are depleted. The method of immortalization is not critical.

Currently, the most common method is fusion with a melanoma fusion partner. Other Techniques include EBV transformation, naked DNA, e.g. oncogenes, retroviruses, etc. for the maintenance of stable cell lines and production of monoclonal antibodies. including any other method of Human monoclonal antibodies can be prepared using appropriate human fusions. It can be obtained by fusion of a partner and the above *i cell. mouse vs mouse Detailed techniques for preparing monoclonal antibodies are described by Oi and Herze Herzenberg [in single-cell immunology] Selected Methods in Cellular Immunology)” Michelle and Shiigi (eds, ), W. H. Freeman and Co., San Francisco (1 980), pp. 351, 372), the antibodies of the present invention can be used with any immunoglobulin. IgG1. IgG2a, IgG2B, and IgA.

IgG includes IgD, IgE, and IgM, and is generally IgG or IgM. It can be gM.

Once antibodies with appropriate specificity have been prepared, they can be used in a wide variety of immunological assays. The Say method becomes useful for detecting p4003N in biological samples. Obi A large number of competitive and non-competitive protein binding assays have been described in the scientific and patent literature. and a number of such assays have been commercialized.

In carrying out the immunoassays of the invention, biological samples are generally prepared in the same manner. must be processed in advance. Sample preparation depends on the biological sample source be changed. Serum samples are typically obtained by coagulating whole blood according to well-known methods. and separating the supernatant. other biological fluids, e.g. semen , sputum and urine can also be prepared in a conventional manner.

Solid tumors and other tumor samples can generally be prepared by disrupting cells.

Unlike the above method, the immuno-Mi tissue chemical staining method is based on the cell surface in appropriately prepared tissue sections. It can be used to detect O5N's in surfaces.

The following examples are offered by way of illustration and are not intended to be limiting.

Example The following abbreviations were used.

BSA: Bovine serum albumin DEAE: Diethylaminoethyl DMEM: Dulbecco's improved Eaglener @Asahi DMSO: Dimethyl sulfoki Sid DTT: Dithiothreitol ELISA: Enzyme-linked immunosorbent assay FBS: Fetal bovine blood Qing HAT: Hyboxanthin-Aminopterin-Thymidine HF: Hydrogen fluoride HPLCH High Performance Liquid Chromatography HIF: Human Transferrin kD: Kilodalton LAI: Leukocyte aggregation inhibition MAb: Monoclonal antibody MHC: Relative mass of major histocompatibility complex Mr two molecules NAI: Non-agglomeration index OSN: Organ-specific cancer neoantigen PAGE: Polyacrylamide Gel electrophoresis PBL: Peripheral blood → white blood cells PBS: Phosphate buffer solution PMSF: Phenylmethylsulfonyl fluoride RBC: Red blood cells SDS: Sodium dodecylsulfonate TFA: Trifluoroacetic acid TPBS: Tween @ phosphate buffer solution up to 1 diy Eight MCI-869 lung cancer cell line: Small cell carcinoma (MCI-869) is a chemically defined Grown in suspension in medium and supplied by Simms et al. (198 0) Cancer Res, 40: 4356-4363). MCl-1 (6 9t8 cells were treated with RPMI 1640 (Gibco) containing 10% FBS, 0. 013M HEPES buffer (N-2-hydroxyethylpiperazine -N'-2-ethanesulfonic acid content: Boehringer Mannheim GmbH), etc. and 0.2% sodium carbonate (Finscher Scientific), or 3 Serum-free chemically defined medium containing sodium selenate of Xl0-@M (anachemi AC-8496), 10-'M (7) High FO: 2-Chiso 7 C'i Bear H -4001), 10-”M 17-β-estradiol (Sigma E-887 5), 5x/- insulin (bovine) (Connaught Labor atoris), 1°C g/ml galactose (Gibco) and 100 ~5 trg/cd human transferrin (HTf) (Hoechst 0TRE) 04105) and grew by adhering to both plastic and glass rotating culture bottles. Ta. HTf is 4,5d of 511 containing 0.1M sodium citrate before use. IMFeC! ! By adding about 80% of HTflg dissolved in distilled water to 2. Iron load (iron-1 loaded) L/. 5.04 for this mixture g of sodium carbonate was added and the pH was adjusted to 7.4.

The final volume of this solution was 100 - and incubated at 25°C for 3 hours and then at 4°C overnight. did. Used antibiotics regularly: 67.0001. U, /l penicillin, 6 7.000■/It strebtomycin (Flow Labs), 500g/ j’Fungizone (FlowLabs) and 30 membranes g/l gentamicin reagent solution (Schering).

Once MCI-H69 becomes confluent, the medium containing the above FBS were isolated and the cells were soaked in phosphate buffer solution (0.14M NaCl, 0.01M phosphate). After washing once with sodium pt17.3): RPMI 1640 was added. Up Serum-free RPMI 1640 was collected after 4B-72 hours, and 10% FB before repeating this cycle. It took an additional 48 hours. To grow NC)-)169 in chemically defined medium, Collect the pent medium (spent-+aediue+) and add fresh medium for 2 Additions were made every 4-48 hours. 100-phenyl fluoride in the freshly collected supernatant. Add methylsulfonyl (PMSF) (Sigma) and 0.02% sodium azide. I got it. Cell remnants were removed by centrifugation at 5000xg for 30 minutes at 4°C. Then top ultraviolet light using Qmiconcell with PM10 membrane It was concentrated approximately 10 times by filtration and stored at -40°C until use. tissue culture The usual ratio of medium volume to surface area of cultured cells is 1 m! /3aJ. Noriyoshi Typically, spent medium 601 containing 2 g of protein is We began the isolation of tumor antigens when they were harvested from endocytic cells.

Shouting plate to the extended spent HCT-15 and 5W-620y3 intestinal cancer cell lines (American type culture) Collection, Lexington, MA) in HEPES buffer (Boehringer - Mannheim, Dorva 1. Quebec + Canada), 0.2 % Sodium Carbonate (Fisher Scientific, Montreal, Quebec, Canada) and initially 10% NU-serum (Collaboray). RPMI 164 containing Tipu Research, Lexington, MA) 0 (Gibco Laboratories, Burlington, Ontario, Ca. nada). When the cell becomes confluent, it The NU-serum was reduced to 1% at a concentration that maintained good growth. antibiotics regularly [Penicillin (67,0001, tl, #!)] used for streptoma Isin (67,000g/2) (Flow Laboratories, Mississippi ga, 0ntario+Canada); 500g Fungizon (same); Gentamicin (30 g/l) reagent solution (shading, Po1nte C1 Aire, Quebec, Canada)) The above confluent cells were 1 %NO- RPMI 1640 medium containing serum for 2 days and serum-free RPMI 1640 medium The 5th day was repeated alternately.

If the serum-free medium turns yellow before the expiry of 5 days, replace it with fresh serum-free RP. Replaced with MI 1640 medium. Under these conditions, the cells undergo constant proliferation. continued, and the rotating bottles were reused over and over again for many months. serum free cyclo Only the spent medium between the cells was collected for the separation and purification of O5N. new The freshly collected supernatant was treated with 100m phenylmethylsulfonyl fluoride (Sigma Chemical Co., St. Louis, MO) was added. a+ Cell remnant layer 4℃, 30 It was removed by centrifugation at 5000xg for 1 minute. The supernatant was then filtered with a PM 10 membrane. Concentrate approximately 10 times by ultrafiltration in a 2-liter Amicon cell, and Stored at -40°C until use. Medium volume relative to surface area of tissue cultured cells The usual ratio of was 1d to 3alI.

Typically, approximately 50 ff of spent medium containing 2 g of protein is used to remove confluent cells. When collected from 20rrr of ll! We have begun to isolate tumor antigens. for this experiment Leave the cells! 2, 13, 26, 21.

Spent media from 20 and 30 days were collected and separated.

O5N's- -E ship drifting 3-92'' SI 1 Sdan Dl) DEAE anion 6 chromatography goofy : Concentrate the above spent medium to 20 - in an Amicon cell with a stirrer equipped with a PM 10 membrane. Condensed and permeated to initial buffer (0,003M) Lith Acetate pH 8°O). sample, centrifuge at 5000xg for 1 hour at 4°C to remove remaining layers, and further Add 60 ml (5 DEAE (LKB, Fisher Scientific, Montrea 1, Canada) column. The column can be used in a batch method while separating the effluent. The initial buffer you want to elute with, 0.003M)) 0.016 in lis acetate. l Vinegar sodium acetate pH 8.0, then 1.0 M sodium acetate in the same buffer).

After dialysis against PBS, the separated solution was ultrafiltered through an Amicon PM 10 membrane. 5×88a11 Cefacryl S, concentrated to 51 or less by filtration and marked with a scale -200 (Pharmacia, Sweden) column and then 15 w1/hv , with PBS and 0.02% sodium azide. 4 minutes from the column fractions were collected (the high molecular fractions that migrated with the displaced volume of the column were labeled “F150 ”; a protein that elutes slightly before standard BSA elutes. The peak was named “F70”; from where standard ovalbumin is eluted. The protein peak eluting slightly later was named “F44”; The low molecular weight fraction eluting within the region where trypsinogen A and cytochrome C elute is It was named "F25". ).

From the above molecular sieve column The sample was concentrated and permeabilized against 0.1M sodium phosphate buffer (pH 7.0). Then, 5-Blue Sepharose CL-6B (Pharmacia, Mon treaLC Canada). The binding protein was dissolved in 3M thiocyanate. It was eluted with lium.

With h-45? + -600OA solvent supply unit, general-purpose liquid chromatog at 116 Rough injector (with small window (100p) for lsj sample), 72 0 system controller, and an Omniscribe recorder ( Connect with Houston In5trua+ent+Au5tin, Texas) A 450 tunable wavelength detector (used at 2B0nm) and an 8d UV transmission cell Waters Corporation (Milford), consisting of

Mass) equipment was used. 25cm x 4.1m Synchro Pack (Sy nchropak) AX-300 anion exchange column (Synchro+a Inc.

1-inde+ Indiaria) was installed as a monitoring column. Tris ( Hydroxymethyl)aminomethane (Sequenal Grade (Pie) rceChemical Co, Roekford 1llinois)) , and sodium acetate (HPLC Grade (Fisher, Mont. eal)] was distilled three times and then prepared with deionized water, CCl312000 cartridge (llillipore corp, +Bed Biological contaminants were removed by flushing through the tubes (Ford, Mass.). buffer solution 7) After filtering through a Mann filter and degassing for 30 minutes used.

Linear salt gradiente moving from 100% A to 100% B buffer in 60 minutes This was done by Initial buffer A is 0.003M) Lis acetate pH 8.0 and buffer B consists of 0 ．． It contained 1M sodium acetate. The flow rate was ld/mtn. The force Before applying the sample to the BRL microdialysis machine (Bethesda Re5 search Lab-ratories, Inc., Gaithersburg JD) against 100-200 volumes of starting buffer and filtered onto a 0.2μ filter. It was filtered through a filter.

Protein peaks were collected, dialyzed, concentrated, and then tested for antigenic activity.

Cation (S nChro ak CM300) 6 HPLC: Monitoring column and 2 5C111X4.1 Tsuru's Synchropunk Mei 300 (Syn-Chrom Inc. , +Linde, ) ndiana) cation exchange columns were used.

Sodium phosphate (Fisher) and sodium chloride (Baker) as described above. Mixed with water. 30 minute linear salt gradient is 0.01M sodium phosphate (pH 6,0) Initial buffer solution consisting of 100% and 0.01M sodium phosphate 0.5 NaCI inside! A final buffer consisting of 100% of a solution (pH 6.0) with Constructed by B. The test was carried out after setting the flow rate to 1 aff/l1in. protein Peaks were collected, dialyzed, concentrated, and then tested for antigenic activity.

Chromatogoofy 5-chelate Sepharose 6B (Pharmacia, Sweden) to 1■/t By flowing 13.2- of ri ZnClz, the Xn4+ ion coordinates. The capacity was 273. The appearance of Xn''* ions during dissolution is 1M sodium carbonate. was eliminated by adding lithium. Thereafter, 0.5M NaCl was added to the column. Equilibration was made with 0.05M) lith acetate buffer (pH 8.0). 1 ■ Antigen The sample was added to this buffer and the pns,o fraction was collected, followed by 0.5M Elute with NaC1, 0.05M) lith acetate (p) t6.0) and pi(6 ,0 fractions were collected. Hydroxyapatite, HA-Ultrogel (LKB, Sweden) (2 m in column, 0.5M potassium phosphate (pH 6,8) and equilibrated by contacting with 0.005M potassium phosphate (pH 6,8). did. 2.2 samples were run on the gel in the same equilibration buffer, then 0.5 e eluted with a linear gradient to M potassium phosphate (pH 6,8) Con-A Sepharose (Pharmacia) (2mZ) was added to the column at 0.5M N. aC1, 1a+M CaC1z, 1+M MnC! ! 2 and 1mM MgCj! The sample was washed with 0.1 M Tris acetate (pH 7.0) to which Z was added. antigen sample The solution was applied to the column and eluted with this buffer. Then, 1.0Mα-methyl- Elute with D-glucoside, followed by α-methyl-D-glucoside with 3MK (J). eluted with S.D. Molecular sieve HPL (J!, equipped with a monitoring column, and te 0. 3M NaCl, 0.05M sodium phosphate (pH 7.0) with HPLC water A 7.5 wm x 60 cn microvacuum (Microp ak) TSK3000SW (Varian, Pa1o Alto, Califo rnia) column. The flow rate is lra! /@in. sample After dialysis and filtration, 200μ was injected into the above column; the protein peak was collected. The solution was concentrated, dialyzed against PBS cpn 7.3) and then tested.

1 plate of this fish Reverse phase HPLC was carried out using the following two methods. The peak was set at 214na+ Detected with a variable wavelength UV detector.

person 11 Porous Bio-Rad (Bio-Rad Hi-Pore) RP31B monitoring collar Mu, 4, 6 X 30 n+ (Bio-Rad Laboratoris, Ri chmond, California).

Buffer A: 0.05% aqueous TFA, Buffer B: 0.05% TFA acetate Nitrile, gradient: held at 0% B for 15 minutes, followed by 1%/B By linear gradient up to 60% B in 5h (flow rate is 0.5+a f/sho in).

Dantate base Bio-Rad Hi-Pore RP31B monitoring column, 4.6 x 250 m. Buffer A: 0.05% aqueous TFA S buffer B: 0.05% TFA Acetonitrile solution, daradient: Flow rate was set to lal/sin, and the flow rate was set to 0 for 15 minutes. %B, followed by a linear graph up to 60%B at 1%/akin. depending on the agent (flow rate is 1 tar/1 Ikin).

'O8N's Hi'na＼ The colonic O5N active substance is Trisacryl-DEAE (F isher 5cienttfic), Sephacryl ) S-200 (Pharmacia), and Blue Sepharose CL-68 (Pharmacia) chromatography as described above. Af Purified p40 undergoes hydrophobic interaction with hydroxyapatite (hydr ophobic 1 interaction) HPLC chromatography I got it. 100 x 7.5 w Bio-Gem hydroxyapatite column ( Bio-Rad Laboratories, Mississauga, 0nt ario, Canada) was equipped as a monitoring column. Initial buffer A is 0 ．． Composed of 10+aM sodium phosphate (pH 6,8) with 3mM CaC1z. Buffer B is 0.5M sodium phosphate with 0.006o+M CaClz added. Consists of thorium (pH 6,8). 100% Buffer A for 25 minutes A linear salt gradient was formed moving into Buffer B. The flow velocity is laf /min.

250X 4.1 tar Synchropafucoprovir hydrophobic interaction column (Te rocbea+ Laboratories+Ltd,, Rexdale, On tario+Canada) was equipped as a monitoring column. 100 in 30 minutes A linear salt gradient moving from % buffer A to 100% buffer B has the form accomplished. Initial buffer A was added with 1.8M ammonium sulfate (Bio-Rad). Buffer B consisted of 0.02M potassium phosphate (pH 7.0), and buffer B was 2% 1 -0.02M potassium phosphate (p)t with butanol 7. Consists of O) . The flow rate was led/akin, and the sample was (Bethesda Re5search Laboratories+ Inc. 100-200 volumes of starting buffer in + Gai Thersburg* MD) and then dialyzed against a 0.2 m cellulose filter (OE 66) (An apec; Ann Arbor, Mich.) and then applied to a column.

Protein peaks were collected, dialyzed, and tested for p40 and O5N activity. salts was prepared with triple distilled water and placed in a Nor-ganic cc1512000 cartridge. Millipote Corp, Jedford.

Removal of biological contaminants was carried out by flowing through MA).

The buffer was filtered through a 0.1n Watmann filter and then degassed for 30 min. I used it after that. All chemical reagents should be used whenever possible unless otherwise indicated. It is of the highest quality and is manufactured by Fisher Scientific. Purchased from r.5cien-tific, Montreal, Canada) φ゛-and carboxymethylated and 40μm colon made All cleavage reactions involve reduced and carboxymethylated p400SN. It was carried out. Derivatization of cysteine residues in this method is important for subsequent cysteine residues. simplifies protein conversion by preventing trimerization of the protein. Next experimental means was used.

100++M tris chloride containing 6M guanidine hydrochloride D of 10111 for lung or colon p40 (100 μ) in A TT (50 μ/m! in water) solution was added. After standing at room temperature for 18 hours, add iodized vinegar. Add acid (20 μl in 20 μl of water) and stir the mixture for 30 min. The reaction was carried out at ℃. Next, add 20 μ of iodoacetic acid made of steel in the same manner, and 2. The 0 reaction solution reacted for 5 hours is treated by pouring the solution into a reverse phase HPLC column. It was desalted by (method A above).

Mbaku law The above carboxymethylated lung or colon p40 antigen was detected by six different methods as follows. I cut it like this: 1. Sprinkle water onto dish 1 and add 50ill of 0.01N hydrochloric acid to 50 CI'l p. A solution of 4o antigen was heated to 100°C in a sealed test tube. After 2 hours, the mixture It was poured onto a reverse phase HPLC column and processed (Method A).

Alternatively, the hydrolysis may be performed by standing in a 75% acetic acid solution at 37°C for 48 hours. The test was carried out by adding lung CM p40.

21. Izumibe Shikoe 2zune Fyu 100 CM p40 antigen with 204 cyanogen bromide solution (300 μ/- in acetic acid) and allowed to stand at 23° C. for 70 hours. this The mixture was applied directly to a reverse phase nptc column (Method B).

3. Tributophin'>, 50ag CM p40 antigen was oxidized for 4.9d. solution (consisting of 300d glacial acetic acid, 150d hydrochloric acid and 40d DMSO). Ta. The reaction was carried out for 2 hours at 4°C for 4.4i1! suppressed with ice-cold ammonium hydroxide; Then 40d cyanogen bromide (300 µ/rs! in 60% formic acid) was added. this reaction was continued for 44 hours at 4°C and then applied to a reversed phase HPLC column (Method B). .

4. Kamiuni no Ekodan. First, the above proteins are degraded in a limited manner by arginine residues. The protein was acylated by the following method. 200a+M containing 6H guanidine hydrochloride A solution of 200 nCM p4o antigen in borate buffer (pti 8.6; 15 0. cef) was added to 4I citraconic anhydride in three portions. Add each and finish After that, the pH of the solution was adjusted to 8-8.5 with 8N NaOH. 3 to room temperature After standing for an hour, the mixture was mixed with 50+wM carbonate containing 20% isopropanol. Desalting on a Bio-Rad TSK125 column in ammonium (pH 8,1) did. Dry the fractions containing the product in a Speed-Vac. Then, trypsin (immobilized on agarose, 3.5 units) and 50 The reaction was carried out in mM ammonium carbonate (pH 8.1) at 37°C. After 23 hours, the The enzyme was isolated by filtration and the acyl protecting group was hydrolyzed in 10% acetic acid. . The mixture was concentrated and processed onto a reverse phase HPLC column (Method A). If cleavage at lysine and arginine is desired, the above induction method The conic anhydride step is omitted.

5. ``Kimoto Pushin''. 50 d of 200mM ammonium carbonate n CM p40 solution (pH 8,1) was treated with 8.8 units (7) TLCK α-Chymotrypsin (immobilized on agarose) was added. Mixed solution for 8 hours37 After reacting at (Method B).

6, pj [water ffl*dissolve the above CM p40 in 500 d anhydrous liquid OF, Then, it was left to stand at room temperature for 3 hours. Removal of BP under fjIi pressure leads to deglycosylation The remaining protein remained.

Mouse feeling and vibe l dormer second 10 emulsified with the same volume of complete Freund's adjuvant in RBALB/c mice. 0 ■Concentrated OSN was injected intraperitoneally (f, P,). Three days before fusion, the mice were given A t, P injection of 50 g IItM thick OSN was made.

Equal numbers of mouse spleen cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Littl Improved efield (1964) Science, 45: 709-710. using 50% (w/v) polyethylene glycol (Mrl, 500) in and fused with mouse melanoma. The above cells were grown in HAT-DMEM selection medium (Ko hler and Milstein (1975) Nature 256: 49 5,497). The surviving hybrid clone is Handley VP 101 plate (Vand P 5c) was used for membrane staining according to the method of et al. O5N Antigen production was determined by ELISA using isolates and live NCl-H69Elf cells. Immunol, Methods 54: 291 296 (1 982)) - Positive cultures were transferred to 96-well plates) (Costar*Ca I'1cke by colonizing the bridge, Mass. Cloned by the limiting dilution method of arn (Kennett et al., Monoclon al Antibodies, P, 374゜P1enua+ Press, Ne w York (1980)) Three fusions were performed.

Eight hybridoma lines were successfully cloned. The hybridoma line has 5% Stored in DJ'IEM with 10% FBS at 37°C in a CO8 moderated incubator. It existed. Hybridoma IgG was extracted from the supernatant using protein A-Sepharose. (Pharmaeia) and also from ascites. and purified by running through anion exchange chromatography. these The cell-line produced antibody is a cyanogen bromide fragment of OSN p40, as detailed below. was used for the Western analysis method.

■Desire 11 certificate Heparinized venous blood from patients with cancer or diseases unrelated to cancer. Buffy Coat (buffy coat) white blood cells (PBL) were separated and as described above. Red blood cells (RBC) were lysed.

In all experiments, IX10'PBL was first added to 0.5 af medium 199. 2,5X10-'M PEG, incubated for 5 minutes at 20°C, and It was then diluted to 199 lXl0' cells/medium. PBL in advance with PGE , to be incubated with patients affected by large tumors or following surgical procedures. White blood cells from the patient do not aggregate and also do not react in the LAI assay. This is because there is a tendency. pcEz is the aggregation property of white blood cells compared to that of a normal test sample. to recover. Leukocytes from control specimens were treated similarly.

゛-LAI Afsey Todoroki “1 out W” As already disclosed in Grosser and Thoa+son The test tube LAI Afusei is a 20af capacity, 16 x 150tm glass test tube. It was carried out in three ways (Cancer Res, 35: 2571-2579) (1975)). Medium 199 (0.3 mf), 0.1 - cancer extract, e.g. lung (=100g protein), and 0. I WL! of suspended leukocytes in 3 assays. Added to test tube. Another 3 test tubes contain 0.3 aff medium 199.0.1- of cancer extract, such as pancreas, and 0.1 d of suspended leukocytes were added. above test Lay the test tube flat and incubate at 37°C in a humidified atmosphere with 5% COt. did.

After 2 hours, stand the test tube upright and pipette the contents at the bottom with a Pasteur pipette. vortex, and then place a sample of non-adherent cells from each tube into a hemocytometer. Then, it was calculated by Convenitor-driven video analysis. connect the computer The device calculates the number of non-agglutinated cells and converts it to the non-agglutinated index ( NAI) results were displayed: NAI=((A-B)/B)X100, where A is equal to the number of non-aggregated cells in the sample after incubation with the initial cancer extract. and B is equal to non-aggregated cells in the sample after incubation with control cancer extract. .

In the above experiment, white blood cells from more than 95% of non-cancer test samples were 30 or less; The standard showed an NAI distributed around 0.

On the other hand, white blood cells from most patients with early-stage cancer are found in test tube A. Despite being similar to that of the sample, it showed an NAI of 30 or more.

Gop'+F=LI: By the improved method of Afusei, the above PEP' of p40 lung cancer OSN The ability of p40 lung cancer O5N to inhibit the aggregation of white blood cells to glass Compared to that which prevents collection. 0.3-medium 199, 0.1 +dc=xo o) control splenic carcinoma extract, 0.1-1×10” suspended leukocytes, and 0.01 mj p40 or peptide diluted appropriately in medium 199 and added to test tube 3. added. 0.31111! The above lungs with medium 199.0.1 d (=100 ag) Cancer extract and 0.1- suspended leukocytes were added in another 3 tests.

Here A is in the sample after incubation with p40 or its peptides. equal to the number of nonadherent cells found in B and B contains p40 or its peptides. Same as above, except that it is equal to the number of non-adherent cells found in samples that do not have Similarly, the non-adherent cells after 2 hours of culture were counted by video analysis, and the results were calculated. be. In another experiment, it was pre-diluted to 1% in medium 199! 00gf of FBS was replaced with a control cancer extract with the same results.

Wang Gongdan 1 plate 1” Moon MiM, L The assay was performed as described above, and the conditions for this assay were as described above. p40 at the temperature that gives the peak response (0,75 irg/tube). Alternatively, peptide was added to tube A. For LAN addition of other substances to test tube A We evaluated the impact of The peak response to p40 was determined by adding additives to p40. compared with the response.

Go1wojiL5 Peptides were prepared using the Merrifield solid phase method on 4-methylbenzhydrylamine resin. and synthesized it. The product is cleaved from the resin and the protecting groups removed using liquid hydrogen fluoride. and left. Each peptide was converted to an unmodified compound (C-terminally carboxamide and N acetate at the end), and three amino acids at the N-terminus (cystine-glycine). Both were synthesized with the addition of lysine). Maleimide the above added peptide -benzoyl-N-hydroxysuccinimide (MBS) or N-succinimidyl Keyhole through its N-terminal cysteine via either bromoacetate Binded to Keyhole limpet hemocyanin. This conclusion The synthetic peptide was used to immunize mice and rats through standard procedures. .

Poetry-come 40 peptides of HPLCl! Increased amount of peptides Figures 1-6 show reversed-phase HPLC peptide maps of lung p40 obtained by various digestion methods. Indicated. Each method produces a set of peptide fragments that differ according to its selectivity. . All of these methods produce large fragments due to the nature of the residues they degrade. For example, cyanogen bromide is degraded by methionine (except for chymotrypsin degradation). , limited acid hydrolysis breaks down the aspartate-proline bond and Syn is mainly broken down into aromatic residues. Anhydrous IP left the peptide backbone intact The carbohydrate component is cleaved from the protein as it is. Looking at the amino acid composition From this, we could roughly estimate the number of fragments expected from each decomposition method.

Peptide fragments from cyanogen bromide (CNBr) degradation were characterized in detail. the The main fragments were analyzed for their amino acid composition and amino acid sequence. these minutes The analysis was carried out by the Protein Analysis Laboratory. tory, 5cripps C11nic) and Research Foundation (Research Foundation, La Jolla, California) It was. Table 1 shows the amino acid composition of lung CM p40 and its seven main CNBr fragments. This is a list. A peak of 30 was observed in the HPLC peptide map. table 2 is a sequence analysis of these five cyanogen bromide (CN) fragments as well as four additions. A typical chymotryptic (CH) fragment is shown.

dog--L CN-2: (N) NTFNNETILHG[)AXQ (C) CN-6: (N) GTFLNFXtlLN GKXXz (C) CN-10= (N) LNKKKGGKEV NKEV (C) CH-13: (N) NNEANE FMEA L (C) CM-18: (N) AAWK (C) CH-22: (N) INENAKDN (C) CH-29: (N) IVTNPIPY (C)X = unknown X, = T or G xz = N or A CNBr fragments of lung p40 were analyzed by 5O5-PAGE and nitrocell. The mixture was transferred to a loin and probed with an antibody against the raw protein. Table these results. It was summarized in 3. The 5DPAGH result yields the molecular weight (and purity) of the fragment. , and the Western method relies on the presence of antigenic determinants in fragments of the source protein. shows. In the Western method, the above CN-10 and CN-12 fragments are whole lung OSN. by two or three monoclonal antibodies (A, B, and C) raised against p40. It was shown that it is recognized as

stolen knee branch The above p40 cyanogen bromide peptide fragments were separated on a 16% acrylamide SDS gel. , then blotted onto nitrocellulose and the above 3. Immunoreaction was performed with a monoclonal antibody.

and its 400SN, its CNBr　'', its HF　'', its top 1psi Boat boat White blood cells from patients affected by lung cancer were tested against different concentrations of intact p4005N. It was tested. Its usage-response curve was narrow and bell-shaped (see Figure 7). , the values in Figure 7 are the arithmetic mean of at least 5 different test samples tested in duplicate. be. The antigen concentration at 0 is the same in tube A and test B without p400SN. NAI values for particle extracts are shown.

The value of >30 is significantly different from the value of 0 (p<0.001). same white blood cells The responses to lung cancer extract in test tube A and pancreatic cancer extract in test tube B are shown on the right. Ta. The peak response had a p40 of 0.5-0.75 per test back. got cancer White blood cells from subjects with no or malignant melanoma were similar to those shown in Figure 7. Not only for higher or lower concentrations, but also for the same concentration. does not respond in any way. CNBr, HF or trypsin digestion of p40 showed similar dose-response curves and peaking responses (Tables 4.5 and 6). child The results show that the above antigenic epitope of p40 is not digested by CNBr degradation, tryptic digestion, or not destroyed by HF degradation and the epitope is restricted in helper T cells. If the determinant is a linear determinant recognized by class-II, It was shown that this is not the case.

Table - 20 NBr''''40 hemorrhoids O3N, white LAI month''b revised I Lung cancer 8±6 42±1039±1361±1114±8 Malignant 2±40±57± 113±85±3 melanoma Control 6 ± 3-9 ± 5-5 Sat 67 ± 104 ± 4 subjects shell i zato Lung cancer 3±4 22±1156±8 66±10 -4±2 Malignant -12±3- 3±70±51±4 melanoma Control 3±52±50±8-2±6- Subject Lung cancer 8.0±11 7.0±1085±1774±1834±15-3±7 bad Sex: 3±50±96±52±41±8-Melanoma Control 11±4-2±810±13-6±3-3±7-Subject As shown above, p40 lung cancer CNBr fragments were separated by reverse phase 1 (PLC). 0 individual peaks were tested by high-dose inhibition. Two different preparations were prepared. manufactured, sampled, and tested.

Peak 8 in the first preparation showed high-dose inhibition and was tested as an antigen. It showed a positive peak response at about 0.2-0.25. second preparation Peak 15 in the sample indicates high-dose inhibition and when tested as an antigen A positive peak response was shown at 0.25n. Peaks 8 and 15 are shown in Table 1゜2 and above. It corresponds to peptide CN-8 described in 3.

Based on these results, the first preparation, peak 8 (CN-8) peptide The sequence of CN-8 was determined, and the sequence corresponding to that of CN-8 was Two corresponding synthetic peptides were prepared. The sequence of the composite was as follows: Peptide 1: FINLAKNKQD TYGPL Peptide 2: TNPIP YDVGV FRKSVNQL Furthermore, both peptides are linked to the amino acid terminus. was prepared with a combined cysteine-glycine-glycine sequence.

As mentioned above, such adducts form immunogens that can be used for the preparation of antibodies. Keyhole limpet used to conjugate the peptide to hemocyanin did.

The above synthetic peptide, named peptide 1, was extensively tested in LAI assays. I tested it. synthetic peptides on white blood cells from lung or breast cancer patients and control subjects. The dose-response tested for Tide 1 is shown in Table 7. White blood cells from lung cancer patients are Peptide 1 showed a positive peak response at 0.175-0.225.

White blood cells from control subjects or breast cancer patients did not react. Test with peptide 1 The responses of different cancer patients to LAN are shown in Table 8. Positive responses came from lung cancer patients It was only found when using white blood cells. Some patients with breast cancer are more likely to have breast cancer than controls. Although high responses were shown, the responses were markedly different from those of lung cancer patients.

Meeting-7: LAI Asse Shinsato Isokozu Petit Shi Danjiri i Niba Sake Trial 2 Lung cancer -14±6-5±324±1344±847±14 birds±616±5-9±1 1 Breast cancer 4±12±51±4-2±79±30±2-15±4-18±9 Control 10±13-4±70±10-2±5-4±82±104±3-11±1 Lung cancer 1046±5 Control 11-4±3 Colon cancer 5-4. ±8 Malignant melanoma 1 -10±10 Bladder cancer 2 -8±2 Breast cancer 69±6 Further coded samples were tested to confirm the specificity of Peptide 1. did. The results are shown in Table 9-14.

Tryptic digest of p40 lung cancer or colon cancer was specifically recognized (No. 9)0 Table 1 0-14 was tested either as an antigen or by high-dose inhibition. We showed that putide 1 elicited responses only in leukocytes from lung cancer patients.

U: 户 ink 蟇瀘 Jl raccoon o λW 脩H gloss 9 trib direct criticism ■ Koni from the other country `` l Koshi friend trial run Colon cancer (N=3) 5±7 35±9 Lung cancer (N-3) 35±215±7 ＜　o, oi　＜　o, os *Code: A=p40 lung cancer; B-p40 colon cancer.

Colon cancer (N=2) 35±21 0±11 7±9 Lung cancer (N=2) 11±21 11±1145±6 Effectiveness NS NS <0.01 *Code: A=T1B peptide; B-control protein: C=synthetic lung peptide , NS = no validity.

Effectiveness NS NS <0.01 *Code = A = T18 peptide, B = control peptide; C = synthetic lung peptide, N S = No effectiveness.

Colon cancer (N=5) -3±1515±633 Sat 942±12 Lung cancer (N=5) 4 0±951±1032±761±14 Effectiveness < 0.001 < 0.01 NS NS* Code: A = Synthetic lung peptide; B = Synthesis of MBP T18°NS = No effectiveness.

Effectiveness <0.001 <0.05 <0.005 <0.05* Code: A = Lung synthetic peptide 1; B = p40 colon tryptic peptide (number of residues 13) Colon cancer (N=3) 53±8 44±8-12±3-14±5 Lung cancer (N=3)- 5±4-10±847±2139±11 Effectiveness < 0.001 < 0.00 5　＜　0.05　＜　0.005*　Code: A=p40 colonic trypsin quenching peptide (number of residues: 13); B = Lung cancer synthetic peptide In conclusion, synthetic peptide 1 has been shown to be effective against the above-mentioned p4 recognized by leukocytes from lung cancer patients. 005N, but is not recognized by leukocytes from patients with other cancers. Not possible.

1μ long 56 y Reversed phase HPLC of colon CM p40 obtained by CNBr and trypsin digestion method77 The major peaks from both digests were sequenced as shown in Figures 8 and 9, respectively. , and the sequence analysis is shown in Table 15 as CCN 8 (N) N-T-F-L-N-V4 -Xi-L-Xs-G (C)B, C(N) Q-5-V-Y-D-P-T-V -Q-A-N (C) B, C (N) V-S-Q-G-Q-W-D-K (C) C, D (N) F-Y-V-D-G-T-D-5-D-P-L-V-K (C ) B, E (N) Y-V-D-G-T-D-5-D-P-L-V-K (C) Trace sequence (N) W-N-G-N-V-T (C) Baa (N) A-5- W-T-D-E-N-) [-K (C) Trace sequence (N) I-D-N-F-Q -K (C) Bza (N) F-T-T-L-N-E-D-R (C) C, b (N) I-F-V-E-T-P-Y (C) Trypsin quenching of colon p4003N Cyanide and cyanogen bromide degradation fragments were characterized in a similar manner to the corresponding lung cancer antigen fragments. It was done.

Both sequence data and LAI activity were obtained for the major fragment.

The most interesting fragments were then prepared by solid phase synthesis.

All major peaks of reversed phase HPLC map in case of cyanogen bromide decomposition Selection was made using the LAI assay by quantitative inhibition. 6 peaks considered positive and these The results of the assay are shown in Table 16. The numbering of the peaks is in the reverse phase) IPLcO diagram. (Figure 8).

Individual activity peaks from the above digests to confirm that their activities are correct. was retested. For example, the above sample of the main active fragment (#10) from cyanogen bromide decomposition The coded test is shown in Table 17.

Trypsin digested fragments were treated similarly. Reverse phase HPLC peptide map (Figure 9) The peaks from The peaks were selected (Table 18). The positive fractions then confirm that their activity is correct. It was screened twice as an antigen for LAI assay to confirm. all activity The fractions were sequenced as described above (Table 15). Main active trypsin digestion Coding tests and dose-response tests for high-dose inhibition of CID are shown in Tables 13 and 14. It was shown to.

Barley: RP-) IPLC't' Attraction gti, dog"" 4 0SN CN Br　”　Gai CCN-6104NT　+ (employed) CCN-138413+ CCN-1614 *HPLC Zamburu fraction is in the colon antigen cyanogen bromide digested peptide map (Figure 8) corresponds to the peak number of

Lung cancer 59±8 (0) 12±8 (103) (n=4) 59±13 Colon cancer 4±4 (96) 51±11 (39) (n=4) 83±22 p < 0.005 p < 0.005 solution B-Synthetic lung bebutide 1 n - number tested *-Table showing that the crude cancer extraction proboscis used was consistent with the diagnosis of the donor leukocytes. ヱLC9 2nd term 15th power Jζ connection J1 cancer cause U- trifushi Y body j bamboo RP-) IP The symbol Lc corresponds to the l-ribsin digested peptide map (column C-3).

C3D 18-14 According to the method described above, the above tryptic fragments C, I) (Table 15) were prepared in the format of 2. Ta.

Peptide 3: (N) FYVDGTDSDP LVK (C) As mentioned above In particular, the adduct is bound to the keyhole green snail hemocyanin and Used for body production.

Dose-response LAI studies for synthetic peptide 3 are shown in Tables 20 and 21. These experiments The results showed that the peptide was recognized by leukocytes from colon cancer patients, but not from lung cancer patients. This indicates that colonic p4o is a determinant of O5N, which is not recognized by leukocytes from other patients. Ru.

Table-4 to 1 cancer peptide 3 Colon cancer in LAI of peptide 52±5 1±9 15±2858±1887±2131±9 Lung cancer 00±15-6±8- 1±75±12-4 soil 81±6* Crude extract is similar to donor leukocytes Lung cancer (n = 3) 11 ± 5 5 soil 4-9 ± 5-4 soil 921 ± 1452 ± 3 glue magazine ( n=3) -4±5-10±538±1445±17-3±47±6NS NS <0.01 <0.05 <0.2 <0.001B = Synthetic colon peptide 3 C=Synthetic lung peptide 1 Antibodies against peptides 1, 2, and 3 were prepared according to the method described above. then , various antisera against each of the peptides were analyzed using an enzyme-linked immunosorbent assay. (ELISA), Western analysis method, and radioimmunoassay (RI A) tested against the peptide as well as against both lung and colon p40. did. The results are shown in Table 22.

Tang's Regarding activity °ζ: +++ = very strong positive ++=strong positive +=positive +/--weak positive One = negative ND = Not implemented The foregoing invention has been described in some detail by way of illustration and example for clarity of understanding. Having thus described, it will be apparent that certain changes and modifications may be practiced within the aspects of the invention. Or maybe.

FIG.

FIG., 2゜ FjG, J.

FIG, 5° Purified lung cancer p40QsN (ug) test tube) extract FIG, -7° FjG, J.

FIG, =IO- international search report It　161　MI　Ml^-”””’　PCT/USA7101595PCT /US87101595 Attachment Forn PCτ/ENGSA/210. Part Engineering.

o, s, CL: 530/324, 326-330; 530/3137, 80 6,828 words! 5earch: CAS 5UBJECT INDEX 9-11TE! C! 0DI from ANT ES AND MONOCLONAL ANT to 0DXES To 0RGA NSPEC Engineering FICNEOANT Engineering GENS.

Claims (21)

[Claims] 1. Leukemia of patients with less than 100 amino acids and affected by solid tumors organ-specific neoantigens that can induce leukocyte aggregation inhibition (LAI) in A peptide fragment that shows immunological cross-reactivity with OSN). 2. Cross-reactive with neoantigens from certain types of tumors, but with all Characterized by virtually no cross-reactivity with neoantigens derived from other types of tumors The peptide fragment according to claim 1. 3. Claim 2, characterized in that it has cross-reactivity with pulmonary neoantigens. Peptide fragment. 4. Characterized by being prepared by proteolysis of the organ-specific neoantigens mentioned above. The peptide fragment according to claim 1, which is 5. It is specially prepared by cyanogen bromide decomposition of the above organ-specific neoantigen. The peptide fragment according to claim 4, wherein the peptide fragment has a characteristic. 6. The peptide fragment according to claim 1, characterized in that it is prepared by chemical synthesis. . 7. characterized in that it is prepared by expression of a recombinant pector in a cell culture host The peptide fragment according to claim 1. 8. It is characterized by cross-reactivity with the epitope on the OSN mentioned above that causes LAI. The peptide according to claim 1, wherein the peptide has a characteristic. 9. At least 80% homology with any of the following amino acid sequences or portions thereof: Peptide fragment having: (I) [Sequence exists]; (II) [There is an array]; (III) [There is an array]; (IV) [There is an array] (V) [There is an array] (VI) [There is an array] (VII) [There is an array] (VIII) [There is an array] (IX) [There is an array] (X) [There is an array] (XI) [There is an array] (XII) [There is an array] (XIII) [There is an array] (XIV) [There is an array] (XV) [There is an array] (XVI) [There is an array] (XVII) [There is an array] (XVIII) [There is an array] (XIX) [There is an array] (XX) [There is an array] (XXI) [There is an array]. 10. Antibodies produced against the peptides according to claim 1. 11. Antibodies produced against the peptides according to claim 9. 12. For detecting the presence of organ-specific neoantigens in biological samples 100 amino acids or less, and patients affected by solid tumors. An organ-specific neoantibody that can induce leukocyte aggregation inhibition (LAI) in human leukocytes. Produced for peptide fragments that have immunological cross-reactivity with OSN The method characterized in that the method comprises exposing the specimen to an antibody. 13. The above peptide fragments are cross-reactive with neoantigens derived from certain types of tumors. but exhibit cross-reactivity with neoantigens from all other types of tumors. 13. The method according to claim 12, characterized in that it does not have any 14. The above peptide fragment crosses with neoantigens derived from at least two types of tumors. 13. A method according to claim 12, characterized in that it is reactive. 15. Claim 12, wherein the fool's sample is a serum sample. How to put it on. 16. Detecting the presence of organ-specific neoantigens in patient blood samples A method for exposing leukocytes from the blood sample to an OSN peptide; and observing inhibition of leukocyte binding as a result of exposure to OSN. Law. 17. The above OSN peptides correspond to various determinant sites on OSNp40. 16. The method according to claim 15, characterized in that it is a mixture of putidos. 18. the OSN peptides are purified to at least about 80% w/w, and according to claim 15, characterized in that it corresponds to a single determinant site on OSNp40. Method. 19. Claim 1, wherein the OSN peptides correspond to lung cancer OSN. 5. The method described in 5. 20. A claim characterized in that the above OSN peptides correspond to colorectal cancer OSN. The method according to claim 15. 21. The above OSN peptides are common to OSN's from at least two types of tumors. 17. The method according to claim 16, characterized in that the method corresponds to a determinant site of.