AU7696487A - Peptide fragments of organ-specific neoantigens - Google Patents

Description

PEPTIDE FRAGMENTS OF ORGAN-SPECIFIC NEOANTIGENS

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the diagnosis of cancer in humans, and more particularly to the preparation of antigenic peptides which are frag- ments of organ-specific tumor antigens capable of mediat¬ ing leukocyte adherence inhibition in the sera of patients suffering from cancer in the specific organ.

With the advent of new and effective methods for cancer therapy, it has become increasingly important to be able to provide early diagnosis and to monitor the status of the cancer during the course of treatment. Many treatment regimens, in order to be effective, must be commenced early in the course of the disease, and it is critical to monitor the tumor load during treatment in order to adjust the treatment regimen accordingly. One approach to the diagnosis and monitoring of cancer is based on the detection of tumor antigens in biological specimens, particularly serum and tissue samples. Such tumor antigens, often referred to as tumor markers, are substances, typically proteins, gly- σoproteins, polysaccharides, and the like, which are produced by tumor cells and characteristic thereof. The tumor antigens may be cell metabolites, cell surface antigens, and the like, typically being cell surface antigens which may be shed into circulation under a variety of circumstances. Detection of such secreted tumor antigens in serum may thus be diagnostic of the malignancy. Although a substantial number of tumor antigens have been identified in the past, no single tumor antigen has thus far provided an entirely reliable basis for cancer diagnosis and monitoring. Therefore, it is de- sirable to identify additional tumor markers which can be used singly or in combination with other tumor markers in the diagnosis and detection of cancer.

A particular 40kD antigen, designated organ- specific neoantigen p40 (OSN p40), has been isolated by Dr. David Thomson's research group at Montreal General

Hospital, Montreal, Quebec, Canada. OSN p40 is expressed on the membranes of solid tumor cells and triggers immuno¬ logically-mediated leukocyte adherence inhibition (LAI) in the peripheral blood leukocytes (PBL) of patients suffering from that type of tumor. While OSN p40 is promising as a tumor marker, its purification is difficult, and its use in leukocyte adherence inhibition assays is limited as antigen -from one type of tumor, e.g., lung, will induce LAI in the sera of patients suffering from two or more types of cancer, e.g., lung and breast as well as lung and bladder.

It would therefore be desirable to provide improved reagents, including antigens and antibodies, capable of specifically detecting OSN p40 from various tumor types in patient specimens. It would be particu¬ larly desirable to provide such reagents which are spe¬ cific to a particular tumor type and free from cross- reactivity with other tumor types.

2. Description of the Prior Art

Extracts from cancer cells inhibit leukocyte adherence in peripheral blood leukocytes from patients with the specific form of cancer (Halliday and Miller (1972) Int. J. Cancer 9:477-483). Tumor antigens from such extracts have been designated as organ-specific cancer neoantigens (Grosser and Thomson (1975) Cancer Res. 35:2571-2579). Organ-specific neoantigens have been shown to be membrane proteins and are not solubilized by crude extraction techniques (Grosser and Thomson

(1979) supra. ) , but may be solubilized by limited papain digestion (Thomson et al. (1976) Cancer Res. 36:3518-3525). At least some organ-specific neoantigens are shed from the cancer cell surface and may be detected in both serum and urine (Grosser and Thomson (1976) Int. J. Cancer 18:58-66). Certain organ-specific neoantigens have been isolated from both serum and urine (Lopez and Thomson (1977) Int. J. Cancer 20:834-848; Thomson et al. (1980) Br. J. Cancer 41:86-99; and Thomson et al.

(1980) Europ. J. Cancer 16:539-551). Tissue cultured cancer cells express the organ-specific neoantigen and shed the neo-antigen into spent medium (Lajzerowicz et al. (1982) J. Urol. 128:1122-1129; and Khosravi et al. (1983) Transplantation 35:258-266). Purification from lung cancer cells of an approximately 40kD antigen re¬ sponsible for inhibition of leukocyte adherence (LAI) in the peripheral blood leukocytes (PBL) of lung cancer patients was described in Thomson et al. (1985) Int. J.

Cancer 35:707-714. A similar antigen capable of inducing LAI in the PBL of colorectal patients has been purified from colon cancer cells. Artigas et al. (1986) Cancer Res. 46: 1874-1881. U.S. Patent No. 4,426,446 describes a method for performing leukocyte adherence inhibition assay characterized by the maximization of cAMP levels in the leukocytes. See also, Thomson et al. (1985) J.

Natl. Cancer Inst. 75:995-1003, and Labateya and Thomson

(1985) J. Natl. Cancer Inst. 75:987-994. SUMMARY OF THE INVENTION The present invention provides methods and compositions useful for the diagnosis and monitoring of various cancers. The compositions include peptide frag- ments having no more than 100 amino acids, usually having 50 or fewer amino acids, which are immunologically cross- reactive with organ-specific neoantigens (OSN's) which are characterized by their ability to induce leukocyte adherence inhibition (LAI) in peripheral blood leukocytes (PBL) of patients suffering from cancer in the tissue of a particular body organ. The OSN's from different body organs are structurally similar and share common framework determinants, but are characterized by one or more unique antigenic determinants which may serve as the basis for distinguishing their cellular origin.

The peptide fragments of the present invention generally include the unique antigenic determinants which are charateristic of cellular origin, but are substantially free from the common framework determinants which are characteristic of two or more tissue types. In some cases, however, it may be desirable to provide peptides embodying the common determinants to allow detection of OSN without regard to the cellular origin of the respon¬ sible tumor. Exemplary peptides capable of specifically inducing LAI in PBL from patients suffering from lung and colon cancer are described in the Experimental sec¬ tion. Peptides which are iπununologically ross-reactive among more than one OSN type have also been prepared. Cancer diagnosis and monitoring is carried out by detecting the OSN's in a biological sample, typ¬ ically serum, urine, or the like. Antibodies to the peptides are provided, and various immunological tech¬ niques may be used for detecting the OSN, such as ELISA, radioimmunoassay, enzyme immunoassay, and the like. By utilizing immunological reagents which are specific for the organ-specific antigenic determinants, the cellular origins of a cancer may be determined. Immunological reagents specific for common antigenic determinants of the OSN will also find use when the cellular origin of the cancer is known. Alternatively, the peptides of the present invention may be used in leukocyte adherence inhibition assays for detecting and diagnosing the pres¬ ence and/or cellular origin of cancer in a patient.

BRIEF DESCRIPTION OF THE DRAWINGS Figs. 1 and 2 are reversed-phase high perfor- mance liquid chromatography (HPLC) maps of peptides generated by limited acid hydrolysis of carboxymethylated lung p40 OSN.

Fig. 3 is a reversed-phase HPLC map of peptides generated by cyanogen bromide cleavage of carboxymethylated lung p40 OSN.

Fig. 4 is a reversed-phase HPLC map of peptides generated by tryptophan cleavage of carboxymethylated lung p40 OSN.

Fig. 5 is a reversed-phase HPLC map of peptides generated by tryptic cleavage at arginine of acylated and carboxymethylated lung p40 OSN.

Fig. 6 is a reversed-phase HPLC map of peptides generated by limited chymotryptic cleavage of carbox¬ ymethylated lung p40 OSN. Fig. 7 is a dose response curve for p40 OSN on leukocytes from patients with lung cancer, malignant melanoma, or benign diseases.

Fig. 8 is a reversed-phase HPLC map of peptides generated by cyanogen bromide cleavage of carboxymethylated colon p40 OSN.

Fig. 9 is a reversed-phase HPLC map of peptides generated by tryptic cleavage of carboxymethylated colon p40 OSN.

Fig. 10 is a reversed-phase HPLC map of peptides generated by tryptic cleavage of reduced and carboxymeth¬ ylated lung p40 OSN. DESCRIPTION OF THE PREFERRED EMBODIMENTS

According to the present invention, antigenic peptide fragments of organ-specific neoantigens (OSN's) capable of inhibiting leukocyte adherence, as described hereinbelow, are utilized in the diagnosis and monitoring of cancer in humans. The OSN's have molecular weights in the range from about 35 to 45 kd, usually in the range from about 37 to 43 kd, usually being about 40 kd and originate from neoplastic or fetal cells from a variety of human body organs, including the lung, colon, breast, bladder, and the like. The OSN's from the dif¬ ferent organs are structurally and immunologically related. By structurally related, it is meant that the amino acid sequences of the various OSN's show at least a 50% homology, usually being at least 60% homology, and often 75% or greater homology. By immunologically related, it is meant that the OSN's from different organs share at least one common epitopic site, usually sharing a plurality of epitopic sites, but usually sharing fewer than all epitopic sites. In the case of OSN's from particular organs, such as lung and breast, however, it is possible that the OSN's will be substantially identical both in structure and in immunological characteristics. The approximately 40 kd OSN's of the present invention will be referred to hereinafter as p40.

Leukocyte adherence inhibition (LAI) is a physiological response mediated by various chemoattract- ants. LAI refers to the ^n vitro inhibition of leukocyte adherence and may be used as the basis for an assay for cellular immunity, as described in U.S. Patent No.

4,426,446, the disclosure of which is incorporated herein by reference. In antigen-induced LAI, the response depends on antigen recognition by sensitized or immune leukocytes. The leukocytes when binding the antigen, release mediators and the mediators inhibit the adherence of about 30% of bystander cells which would otherwise be adherent. The antigen (OSN) recognized by leukocytes from a human tumor host is organ-specific. That is, OSN originating in neoplastic cells from a particular body organ will induce LAI in leukocytes from patients suffering from cancer of that organ, but usually not from cancer of other organs. There is, however, some redundancy observed. For example, OSN from neoplastic lung cells will induce partial LAI in PBL from patients suffering from breast cancer, as well as from lung cancer. Peptide fragments according to the present invention will be either haptenic or antigenic and will include at least 6 amino acids and fewer than 100 amino acids, usually including at least 9 amino acids and fewer than 75 amino acids, more usually including in the range from 12 to 75 amino acids found within a nat- ural OSN. The peptide fragments will embody one or more epitopic sites corresponding to unique antigenic determinants characteristic of the OSN. These epitopic sites will be recognized by leukocytes from cancer pa¬ tients, or by antibodies specific for the epitopic sites, or both. Usually, the epitopic site(s) will correspond to antigenic determinants characteristic of the cellular origin of the OSN (to allow for determination of cellular origin of a solid tumor) , but sometimes it may be de¬ sirable to provide peptides having epitope site(s) cor- responding to antigenic determinants common to two or more OSN's having different cellular origins.

The peptides may be natural, i.e:, fragments of OSN isolated from a natural source, or may be syn¬ thetic. The natural peptides may be isolated from natural sources, such as cell lines known to produce the OSN, by conventional techniques such as affinity chromatography. Conveniently, polyclonal or monoclonal antibodies obtained according to the present invention may be used to prepare a suitable affinity column by well known techniques. Such techniques are taught, for example, in Hudson and Hay, Practical Immunology, Blackwell Scientific Publi¬ cations, Oxford, United Kingdom, 1980, Chapter 8. A specific method for isolating the peptides is set forth in the Experimental section hereinafter. The peptides may then be obtained by chemical or enzymatic cleavage of the intact protein, as described in the Experimental section hereinafter. Because of the difficulty in isolating intact

"OSN's from natural sources, however, it is preferred to produce synthetic fragments based on the sequence of the natural OSN_* portions of which are set forth below for lung and colon OSN. Synthetic polypeptides which are immunologically cross-reactive with the natural OSN may be produced by either of two general approaches. First, polypeptides having fewer than about 60 amino acids, more usually fewer than about 30 amino acids, may be synthesized by the well-known Merrifield solid- phase synthesis method where amino acids are sequentially added to a growing chain bound to a solid phase (Merrifield (1963) J. Am. Che . Soc. 85:2149-2156).

The second method for synthesizing- the peptides of the present invention involves the expression in cultured cells of recombinant DNA molecules encoding a desired portion of the OSN gene. The OSN gene may itself be natural or synthetic with the natural gene obtainable from cDNA or genomic libraries using degenerate probes based upon the known amino acid sequences set forth above. Suitable cDNA and genomic libraries may be ob¬ tained from human cell lines known to contain the N-myc gene, such as the NCI-H69 lung cancer cell line. Al¬ ternatively, polynucleotides may be synthesized by well- known techniques. For example, short single-stranded DNA fragments may be prepared by the phosphoramidite method described by Beaucage and Carruthers (1981) Tett. Letters 22.1859-1862. A double-stranded fragment may then be obtained either by synthesizing the complemen¬ tary strand and annealing the strands together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence. The natural or synthetic DNA fragments coding for a desired OSN fragment will be incorporated in DNA constructs capable of introduction to and expression in ^an iS vitro cell culture. Usually, the DNA constructs will be suitable for replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction and integration within the genome of cul¬ tured mammalian or other eukaryotic cell lines. DNA constructs prepared for introduction into bacteria or yeast will include a replication system recognized by the host, the OSN DNA fragment encoding the desired polypeptide product, transcriptional and translational initiation regulatory sequences joined to the 5'-end of the OSN DNA sequence, and transcriptional and trans- lational termination regulatory sequences joined to the 3'-end of the OSN sequence. The transcriptional regu¬ latory sequences will include a heterologous promoter which is recognized by the host. Conveniently,^' avail¬ able expression vectors which include the replication system and transcriptional and translational regulatory sequences together with an insertion site for the OSN DNA sequence may be employed.

As reported in greater detail in the Experi¬ mental section hereinafter, 21 particular peptides having from 4 to 55 amino acids corresponding to lung and colon OSN have been prepared from natural sources. The se¬ quences of these polypeptides (using the standard single letter designation) are as follows:

(I) GTFLNFX-^LN G XX₂

(II) EALIVTNPIP YDVOVFRKSV NQLEQNLIVQ TFINLAKNKQ DTYGPLTGYN GYKXI

(III) LNKKKGGKEV NKEV

(IV) LSKKKGGKEV NKEVTNTFLE NFXLEN

(V ) LNKKKGGKEV NKEWNTFLE NXK

(VI) NNEANEFMEA L

(VII) AAWK

(VIII) INENAKDN

(IX) IVTNPIPY

(X) LNKKKGGKEV NKEVSNTXX₃E NXKGG

(XI) NTFNNETILH GKDAXQ

(XII) NTFLNVTX₄LX₅ G

(XIII) QSVYDPTVQA N

(XIV) VSQGQWDK

(XV) FYVDGTDSDP LVK

(XVI) YVDGTDSDPL VK

(XVII) WNGNVT

(XVIII) ASWTDENHK

(XIX) IDNFQK (XX) FTTLNEDR

(XXI ) IFVETPY

Of these 21, it has been found that peptides III, IV,

V, and X have strong reactivity with anti-p40 monoclonal antibodies directed toward a framework determinant of the native protein. Peptide II contains a determinant of the lung p40 OSN which is recognized by leukocytes from lung cancer patients but not by leukocytes from patients with other cancers. As described in the Exper¬ imental section, synthetic fragments of peptide II have been prepared and demostrate the same activity.

A peptide of 12 amino acids derived from colon p40 OSN has also been isolated (corresponding to peptide XV above), as described in the Experimental section. The peptide was sequenced, and a synthetic peptide pre¬ pared based on that sequence. The synthetic colon pep¬ tide had the following sequence: FYVDGTDSDP LVK

The synthetic peptide was used to prepare antibodes which reacted positively with both colon and lung p40, as well as very strongly positive with the peptide itself. The peptide fragments will usually have at least about 80% homology with the natural amino acid sequence of the OSN, more usually having at least 90% homology, and frequently being identical in sequence. Limited substitutions of amino acids may be made, par¬ ticularly when the substitutions do not affect the an- tigenic properties of the peptides. The peptides may be modified at one or more sites to enhance their prop¬ erties. This includes inserting, deleting, or modifying one or more amino acids or groups. The peptide may be appended with other amino acids or groups to facilitate conjugation to proteins, other peptides, polyamino acids, solid supports or small molecules such as chromophores or fluorophores. The peptide may be appended with groups to modulate its overall hydrophilicity. For example, hydrophobic amino acids or fatty acids such as palmitic acid could be added to promote peptide-containing micelle formation. The amino terminanl may be free or acylated and the carboxy terminal may be the free acid or car- boxamide. Also, the peptides may be joined to other amino acid sequences, such as immunogens, useful for particular purposes.

To be useful in the detection methods of the present invention, the peptides are usually obtained in substantially pure form, that is, typically about 50% w/w or more purity, substantially free of interfering proteins and contaminants. Preferably, the OSN peptides are isolated or synthesized in a purity of at least about 80% w w and, more preferably, in at least about

95% w/w purity. Using conventional protein purification techniques, homogeneous peptides of at least 99% w/w can be obtained. For example, the proteins may be puri¬ fied by use of the antibodies described hereinafter using immunoadsorbent affinity chromatography. Such affinity chromatography is performed by first linking the antibodies to the solid support and then contacting - the linked antibodies with the source of the OSN proteins, e.g., lysates of cells which naturally produce OSN or which produce OSN as a result of introduction of a recom- binant OSN DNA molecule.

The OSN peptides of the present invention are used in the detection and monitoring of neoplastic diseases, particular solid tumors, such as lung cancers, colorectal cancers, breast cancers, prostate cancers, hepatic cancers, and the like. The detection and monitoring are performed by assaying a suitable biological specimen, usually by LAI assays or by known immunological assays. LAI assays may be carried out as described in U.S. Patent No. 4,426,446, previously incorporated by reference. Immu¬ nological techniques either employ the peptide as an antigen for detecting antibody in patient sera, or rely on the preparation of antibodies from the peptides to allow for detection of antigen in a wide variety of biological specimens.

The use of the peptides in LAI assays is sim- ilar to the use of intact p40 OSN, as described in detail in the Experimental section hereinafter. The LAI assay may be performed with a peptide mixture, e.g., a cyanogen bromide cleavage mixture of the native p40 OSN. Alter¬ natively, the assay may be performed with purified pep- tides, either natural or synthetic.

Antibodies to p40 OSN peptides may be obtained by injecting the purified peptide into a wide variety of vertebrates in accordance with conventional techniques. Suitable vertebrates include mice, rats, sheep, and goats, and in particular mice." Usually, the animals are bled periodically with successive bleeds having improved 'titer and specificity. The antigens may be injected intramuscularly, intraperitoneally, subcutane- ously, or the like. Usually, a vehicle is employed, such as a complete or incomplete Freund's adjuvant. If desired, monoclonal antibodies can be prepared.

To obtain monoclonal antibodies, spleen cells from the immunized vertebrate are immortalized. The manner of immortalization is not critical. Presently, the most common method is fusion with a myeloma fusion partner. Other techniques include EBV transformation, transformation with bare DNA, e.g., oncogenes, retro- viruses, etc., or any other method which provides for stable maintenance of the cell line and production of monoclonal antibodies. Human monoclonal antibodies may be obtained by fusion of the spleen cells with an ap¬ propriate human fusion partner. A detailed technique for producing mouse x mouse monoclonal antibodies is taught by Oi and Herzenberg, in "Selected Methods in Cellular Immunology," Mishell and Shiigi (eds.), W. H. Freeman and Co., San Francisco (1980), pp. 351-372. The antibodies of the present invention may be of any -immunoglobulin class, i.e, IgG, including IgGl, IgG2A, and IgG2B, IgA, IgD, IgΞ, and IgM, usually being IgG or IgM.

Once antibodies having suitable specificity have been prepared, a wide variety of immunological assay methods are available for detecting the p40 OSN in a biological sample. Numerous competitive and non- competitive protein binding assays have been described in the scientific and patent literature, and a large number of such assays are commercially available.

In performing immunoassays according to the present invention, it will usually be necessary to pre- treat the biological sample in some manner. Sample preparation will vary depending on the source of the biological sample. Serum samples will typically be prepared by clotting whole blood and isolating the super- natants in accordance with well known methods. Other biological fluids, such as semen, sputem, and urine, may also be prepared by conventional techniques. Solid tumors and other tissue samples will usually be prepared by lysing the cells. Alternatively, immunohistochemical staining may be used to detect the OSN's on the cell surface in properly prepared tissue sections.

The following examples are offered by way of illustration, not by way of limitation.

EXPERIMENTAL The following abbreviations are employed. BSA: bovine serum albumin

DEAE diethylaminoethyl DMEM Dulbecco's Modified Eagle's medium

DMSO dimethylsulfoxide

DTT: dithiothreitol ELISA: enzyme linked immunosorbent assay

FBS: fetal bovine serum

HAT: hypoxanthine-aminopterin-thymidine

HF: hydrogen fluoride

HPLC: high pressure liquid chromatography

HIF: human transferrin kD: kilodalton

LAI: leukocyte adherence inhibition

MAb: monoclonal antibody

MHC: major histocompatibility complex

Mr: relative molecular mass

NAI: nonadherence index

OSN: organ-specific cancer neoantigen

PAGE: polyacrylamide gel electrophoresis

PBL: peripheral blood leukocytes

PBS: phosphate buffered saline

PMSF: phenylmethylsulfonyl fluoride

RBC: red blood cells

SDS: sodium dodecyl sulfate

TFA: trifluoroacetic acid

TLCK: N-α-p-tosyl-L-lysine chloromethyl ketone

® TPBS: Tween phosphate buffered saline

Materials and Methods

Lung Cell Structure and Harvesting of Spent Medium NCI-H69 Lung Cancer Cell Line: A small cell type lung carcinoma, NCI-H69, grows in suspension in chemically- defined medium and was provided by Simms et al. ((1980) Cancer Res. 40:4356-4363). NCI-H69 cells were grown adherent to both plastic and glass roller culture bottles in RPMI 1640 (Gibco) containing 10% FBS, 0.013M Hepes buffer (Boehringer Mannheim GmbH), and 0.2% sodium bi¬ carbonate (Fisher Scientific), or chemically-defined serum-free medium containing 3 x 10 —8M sodium selenite g

(Anachemia AC-8496), 10 M hydrocortisone (Sigma H-4001), 10^~8M 17-β-estradiol (Sigma E-8875), 5 ug/ml insulin (bovine) (a gift from Connaught Laboratories), 1 mg/ml galactose (Gibco) and 100 to 5 ug/ml human transferrin (HTf) (Hoechst OTRE 04/05). HTf was iron-loaded before use by adding one gram of HTf dissolved in approximately 80 mis of distilled water to 4.5 mis of 5mM FeCl, in 0.1M sodium citrate. To this mixture, 5.04 grams of sodium bicarbonate were added and the pH was adjusted to 7.4. The final volume of the solution was brought to 100 mis and was incubated for 3 hrs at 25°C and then overnight at 4°C. Antibiotics were used routinely: 67,000 I.U./L Penicillin, 67,000 ug/L Streptomycin (Flow Labs), 500 ug/L Fungizone (Flow Labs) and 30 ug/L Gen- tamycin Reagent Solution (Schering).

Once NCI-H69 had reached confluency, the medium containing FBS was removed, the cells were washed once with phosphate buffered saline (0.14M NaCl, 0.01M Na Phosphate pH 7.3) (PBS), an then RPMI 1640 was added. The serum-free RPMI 1640 was harvested after 48 to 72 hrs and replaced with RPMI 1640 containing 10% FBS for another 48 hrs before repeating the cycle. For NCI-H69 grown in chemically-defined medium, the spent-medium was harvested and fresh medium was added every 24 to 48 hrs. To freshly harvested supernatants, 100 uM phenyl- methylsulfonyl fluoride (PMSF) (Sigma) and 0.02% sodium azide were added. Cellular debris was removed by centri- fugation at 5000 xg for 30 min at 4°C. Supernatants were then concentrated approximately 10 fold by ultra- filtration in an A icon cell with a PM10 membrane and stored at -40°C until used. The usual ratio of medium volume to surface area of tissue cultured cells was 1

2 ml/3cm . Typically, isolation of the tumor antigen was begun when 60 liters of spent-medium containing 2 gm of

2 protein had been harvested from 20m of confluent cells.

Colon Cell Culture and Harvesting of Spent Media

HCT-15 and SW-620 colon cancer cell lines (Americal Type Culture Collection, Lexington, MA) were grown adherent to plastic or glass roller culture bot¬ tles in RPMI 1640 (Gibco Laboratories, Burlington, Ontario, Canada), containing HEPES buffer (Boehringer Mannheim, Dorval, Quebec, Canada), 0.2% sodium bicar¬ bonate (Fisher Scientific, Montreal, Quebec, Canada), and initially 10% NU-Serum (Collaborative Research, Inc., Lexington, MA). Once the cells were confluent, NU-Serum was decreased to 1% at which concentration it maintained good growth of the cells. Antibiotics were used routinely: penicillin (67,000 IU/liter) ; strepto¬ mycin (67,000 μg/liter) (Flow Laboratories, Mississauga, Ontario, Canada); 500 yg Fungizone (Flow Laboratories); and gentamycin (30 yg/liter); reagent solution (Schering, Pointe Claire, Quebec, Canada). The confluent cells were alternated between 2 days of RPMI 1640 medium con¬ taining 1% NU-Serum and 5 days of serum-free RPMI 1640 medium. When the serum-free medium turned yellow before the end of the fifth day, it was replaced with fresh serum-free RPMI 1640 medium. Under these conditions, the cells maintained constant growth, and the roller bottles were repeatedly recycled over many months.

Only spent medium during the serum-free cycle was harvested for isolation and purification of the OSN. To the freshly harvested supernatants was added 100 yM phenylme hylsulfonyl fluoride (Sigma Chemical Co., St. Louis, MO). Cellular debris was removed by centrifugation at 5000 x g for 30 min at 4°C. Super- natants were then concentrated approximately 10-fold by ultrafiltration in a 2-liter Amicon cell with a PM10 membrane and stored at -40°C until used.

The usual ratio of medium volume to surface

2 area of tissue-cultured cells was 1 ml to 3 cm . Typi- cally, isolation of the tumor antigen was begun when about 50 liters of spent medium containing 2 g of protein

2 had been harvested from 20 m of confluent cells. In this study, spent medium from 12, 13, 26, 21, 20, and

2 30 m of cells was harvested and separated. Physicochemical Separation of Lung OSN

Trisacryl DEAE Anion Exchange Chromatography: The spent medium was concentrated to 20 ml in stirred Amicon cells with PM10 membranes, dialysed against starting buffer (0.003M Tris acetate pH 8.0), centrifuged at 5000 xg for 1 hr at 4°C to remove debris and applied to a 60 ml (5 x 3 cm) Trisacryl DEAE (LKB, Fisher Scientific Montreal, Canada) column-equilibrated in the same buffer. The column was eluted by a batch method with the effluents collected separately. starting buffer, 0.016M sodium acetate in 0.003M Tris acetate pH 8.0 and 1.0M sodium acetate in the same buffer. Sephacryl S-200 Molecular Sieve Chromatography: After dialysis against PBS, the isolates were concentrated to less than 5 mis by ultrafiltration on an Amicon PM10 membrane, applied to a calibrated 5x88 cm Sephacryl S-200 (Pharmacia, Sweden) column and eluted with PBS and 0.02% sodium azide at 75 mls/hr. Four fractions were collected from the column: a high molecular weight fraction, designated "F150", moving with the excluded volume of the column; a protein peak eluting slightly ahead of where a BSA standard eluted, designated "F70"; a protein peak eluting slightly after the area where an ovalbumin standard eluted, designated "F44"; and a low molecular weight fraction eluting in the area where a chymotrypsinogen A and cytochrome C standard eluted, designated "F25". Blue Sepharose CL-6B Chromatography: Samples from the molecular sieve column were concentrated, dialysed against 0.1M sodium phosphate buffer, pH 7.0 and passed through 5 ml of blue Sepharose CL-6B (Pharmacia, Montreal, Canada). Bound proteins were eluted with 3M potassium thiocyanate. Anion (SynChropak AX-300) Exchange Using

High Pressure Liquid Chromatography (HPLC) : A Waters Assoc. (Milford, Mass.) system was used, consisting of an M-45 and an M-6000A solvent delivery unit, a U6K universal liquid chromatograph injector (with a 1 ml sample loop), a 720 system controller, and a 450 variable wavelength detector (used at 280 nm and with an 8 pi UV flow through cell) coupled to an Omniscribe recorder (Houston Instrument, Austin, Texas). The 25 cm x 4.1mm Synchropak AX-300 anion exchange column (Synchrom Inc., Linde, Indiana) was equipped with a guard column. Tris (hydroxymethyl) aminomethane, Sequenal Grade (Pierce Chemical Co., Rockford, Illinois), and sodium acetate, HPLC Grade (Fisher, Montreal), were made up in triple distilled and deionized water, rendered free of organic contaminants by passage through a Norganic CC1512000 cartridge (Millipore Corp., Bedford, Mass.). The buffers were filtered through a 0.1 ym Whatman filter and degassed for 30 minutes before use. Moving from 100% A to 100% buffer B over 60 min, a linear salt gradient was formed. Starting buffer A consisted of 0.003M Tris acetate pH 8.0 and buffer B contained 0.1M sodium acetate in 0.003M Tris acetate pH 8.0. The flow rate was 1 ml/min. Before application to the column, samples were dialysed against 100 to 200 volumes of starting buffer in a BRL Microdi- alysis System (Bethesda Research Laboratories, Inc.,- Gaithersburg, MD) and filtered through a 0.2 ym filter. Protein peaks were collected, dialyzed, concentrated and tested for antigen activity.

Cation (SynChropak CM300) Exchange HPLC: A guard column and a 25 cm x 4.1mm SynChropak CM300 (SynChrom Inc., Linde, Indiana) cation exchange column were used. Sodium phosphate (Fisher) and sodium chloride (Baker) were made up in the water described above. A 30 min linear salt gradient was formed with starting buffer A consist¬ ing of 100% 0.01M sodium phosphate, pH 6.0 and ending with buffer B containing 100% 0.5M NaCl in 0.01M sodium phosphate, pH 6.0/ The flow rate was 1 ml/min before application. Samples were dialyzed and filtered as described above. Protein peaks were collected, dialyzed, concentrated and tested for antigen activity. Other Chromatographic Procedures: Five ml of chelating Sepharose 6B (Pharmacia, Sweden) was charged to 2/3 of its capacity with Zn++ ions by passage of 13.2 mis of 1 mg/ml ZnCl_. Appearance of Zn++ ions in the eluate was excluded by addition of 1M Na Carbonate. The column was then equilibrated with 0.5M NaCl in 0.05M Tris acetate buffer pH 8.0. A 1 mg antigen sample was applied in this buffer, and a pH 8.0 fraction was collected, followed by a pH 6.0 fraction eluted with 0.5M NaCl, 0.05M Tris acetate, pH 6.0. Hydroxylapatite, HA-Ultrogel (LKB, Sweden) (2 mis) was washed in a column with 0.5M K Phos¬ phate pH 6.8 and equilibrated against 0.005M K Phosphate pH 6.8. A 2.2 mg sample was applied in the same e uili- bration buffer to the gel and eluted with a linear gra¬ dient to 0.5M K Phosphate pH 6.8. Con-A Sepharose (Pharmacia) (2 mis) was washed in a column with 0.5M NaCl, lmMCaCl₂, ImM MnCl_2> lmM MgCl₂ in 0.1M Tris ace¬ tate pH 7.0. Antigen samples were applied and eluted with this buffer, then eluted with 1.0, α-methyl-D-glu- coside and followed by 3M KC1 in 1.0M α-methyl-D-glu- coside. Molecular sieve HPLC was performed on a 7.5mm x 60 cm Micropak TSK 3000 SW (Varian, Palo Alto, Cali¬ fornia) column equipped with a guard column and equili- brated with 0.3M NaCl, 0.05M Na Phosphate, pH 7.0 made up in HPLC water. The flow rate was 1 ml/min. Samples of 200 yg were dialyzed, filtered and injected onto the column; protein peaks were collected, concentrated, dialyzed against PBS pH 7.3 and tested. Reversed-Phase HPLC

Reversed-phase HPLC was performed by two methods as set below. Peaks were detected with a variable wave¬ length UV detector set at 214 nm. Method A. Bio-Rad Hi-Pore RP318 Guard Column, 4.6 x 30mm (Bio-Rad Laboratories, Richmond, California) . Buffer A: 0.05% aqueous TFA, Buffer B: 0.05% TFA in acetonitrile. Gradient: Hold at 0% B for 15 minutes followed by a linear gradient to 60% B at one percent per minute, flow rate is 0.5 mL/min. Method B. Bio-Rad Hi-Pore RP318 Guard Column, 4.6 x 250mm Buffer A: 0.05% aqueous TFA, Buffer B: 0.05% TFA in acetonitrile. Gradient: Hold at 0% B for 15 minutes followed by a linear gradient to 60% B at one percent per 1.5 minutes, flow rate is 1 ml/min. Physicochemical Separation of Colon OSN

The colon OSN active materials were separated by Trisacryl-DEAE (Fisher Scientific), Sephacryl S-200 (Pharmacia, Dorval, Quebec, Canada), and blue-Sepharose CL-6B (Pharmacia) chromatography as described above. The affinity-purified p40 was subjected to hydroxylapatite and hydrophobic interaction HPLC chromatography. A 100 x 7.5mm Bio-Gel hydroxylapatite column (Bio-Rad Labora¬ tories, Mississauga, Ontario, Canada) was equipped with a guard column. Starting Buffer A consisted of 0.3 mM CaCl, in 10 mM sodium phosphate, pH 6.8, and Buffer B consisted of 0.006 mM CaCl^ in 0.5M sodium phosphate, _PH 6.8. Moving from 100% Buffer A to 100% Buffer B over 25 min, a linear salt gradient was formed. The flow rate was 1 ml/min.

The 250 x 4.1mm Synchropak propyl hydrophobic interaction column (Terochem Laboratories, Ltd. , Rexdale, Ontario, Canada) was equipped with a guard column.

Moving from 100% Buffer A to 100% Buffer B over 30 min, a linear salt gradient was formed. Starting Buffer A consisted of 1.8M ammonium sulfate (Bio-Rad) in 0.02M potassium phosphate, pH 7.0, and Buffer B consisted of 2% 1-butanol in 0.02M potassium phosphate, pH 7.0. The flow rate was 1 ml/min. Before application to the column, samples were dialyzed against 100 to 200 volumes of starting buffer in a BRL microdialysis system (Bethesda Research Laboratories, Inc., Gaithersburg, MD) and fil- tered through a 0.2mm cellulose filter (0E66) (Anapec, Ann Arbor, MI). Protein peaks were collected, concen¬ trated, dialyzed, and tested for p40 and OSN activity. The salts were prepared in triple distilled water, rendered free of organic contaminants by passage through a Norganic CC1512000 cartridge (Millipore Corp. , Bedford, MA). The buffers were filtered through a 0.1mm Whatman filter and degassed for 30 min before use. All chemicals unless indicated otherwise were of the highest grade possible and purchased from Fisher Scientific (Montreal, Canada). Preparation of Reduced and Carboxymethylated Lung and Colon p40 (CM p40)

All cleavage reactions were performed on re¬ duced and carboxymethylated p40 OSN. Derivatization of the cysteine residues in this manner simplifies further protein transformations by preventing cysteine dimeri- zation. The following procedure was used.

To a solution of lung or colon p40 (100 yg) in 100 mM Tris chloride, pH 8.5 containing 6M guanidine hydrochloride was added 10 yl of a solution of DTT (50 mg/mL in water) . After standing 18 hours at room tem- perature, a solution of iodoacetic acid (20 mg in 20 yl of water) was added, and the mixture was incubated at 37°C for 30 minutes. A second 20 mg aliquot of iodo¬ acetic acid was added and incubation was continued for 2.5 hours. The solution was desalted by applying it directly to a reversed-phase HPLC Column (Method A, above) . Protein Cleavage Methods

The carboxymethylated lung or colon p40 antigen was cleaved by six different methods, as follows: 1. Limited Acid Hydrolysis. A solution of

50 yg of the CM p40 antigen in 50 yl of 0.015 N hydro¬ chloric acid was heated at 100°C in a sealed tube. After 2 hours, the mixture was applied directly to a reversed-phase HPLC column (Method A) . Alternately, the hydrolysis was carried out by allowing the lung CM p40 to stand in a solution of 75% acetic acid at 37°C for 48 hours.

2. Cyanogen Bromide Cleavage. 100 yg of CM p40 antigen was dissolved in 20 yl of a solution of cyanogen bromide (300 mg/ml in acetic acid) and allowed to stand 70 hours at 23°C. The mixture was applied directly to a reversed-phase HPLC column (Method B) . 3. Tryptophan Cleavage. 50 μg of CM p40 antigen was dissolved in 4.9 yl of an oxidation solution (consisting of 300 yl of glacial acetic acid, 150 yl of 9N hydrocloric acid and 40 yl of DMSO) . After 2 hours at 4°C the reaction was quenched with 4.4 yl of ice cold ammonium hydroxide and 40 yl of a cyanogen bromide solution (300 mg/ml in 60% formic acid) was added. This reaction was allowed to proceed for 44 hours at 4°C and then applied directly to a reversed-phase HPLC column (Method B) .

4. Tryptic Cleavage. To restrict cleavage to arginine residues, the protein was first acylated according to the following procedure. To a solution of 200 yg of CM p40 antigen in 200 mM borate buffer con- taining 6M guanidine hydrochloride, pH 8.6 (150 yl) was added three 4 yl aliquots of citraconic anhydride. After each addition, the pH of the solution was adjusted to 8 --8.5 with 8N NaOH. After standing 3.hours at room temperature, the mixture was desalted on a Bio-Rad TSK 125 column in 50 mM ammonium bicarbonate, pH 8.1, containing 20% isopropanol. The product-containing fraction was concentrated to dryness in a Speed-Vac, then incubated at 37°C with trypsin (immobilized on agarose, 3.5 units) in 50 mM ammonium bicarbonate, pH 8.1. After 23 hours the enzyme was removed by filtration and the acyl blocking groups hydrolyzed in 10% acetic acid. The mixture was concentrated and applied directly to a reversed-phase HPLC column (Method A) . If cleavage at lysine and arginine is desired, the derivatization step with citraconic annhydride is omitted.

5. Limited Chymotryptic Cleavage. To a solution of 50 yg CM p40 in 200 yl of 200 mM ammonium bicarbonate, pH 8.1 was added 8.8 units of TLCK-treated α-chymotrypsin (immobilized on agarose). After incubat- ing at 37°C for 8 hours the solution was filtered, ad¬ justed to pH 2.5 and injected directly onto a reversed- phase HPLC Column (Method B) . 6. Hydrogen Fluoride Cleavage. The CM p40 was dissolved in 500 yl of annhydrous liquid HF and allowed to stand at room temperature for 3 hours. Re¬ moval of the HE under vacuum left the deglycosylated protein.

^"Mouse Immunization and Production of Hybridoma Cell Lines

Female BALB/c mice were given intraperitoneal (i.p.) injections of 100 yg of an enriched OSN emulsi- fied in an equal volume of complete Freund's adjuvant. Three days before fusion, the mice were given an i.p. injection of 50 yg of enriched OSN. Equal numbers of mouse splenocytes were fused with the mouse myeloma line, NS-1, with 50% (W/V) polyethylene glycol (Mr 1,500) in Dulbecco's Modified Eagle's medium (DMEM) (method modified from Littlefield (1964) Science 45:709-710). The cells were grown in HAT-DMEM selection medium (Kohler and Milstein (1975)- Nature 256':495-497) . Viable hybrid clones were tested for antibody production by ELISA using the OSN isolate and viable NCI-H69 cells, for membrane staining, using VP 107 plates (V and P Scien¬ tific Inc., San Diego, CA) by the method of Handley et al. (1982) J. Immunol. Methods 54:291-296. Positive cultures were cloned by the limiting dilution method of McKearn in: Kennett et al., Monoclonal Antibodies, p.

374, Plenum Press» New York (1980) , by plating in 96-well plates (Costar, Cambridge,^" Mass.). Three fusions were performed. Eight hybridoma lines were cloned successfully. The hybridoma lines were maintained in DMEM with 10% FBS at 37°C in a 5% C0₂ buffered incubator. The hybridoma IgG was purified from supernatants using a Protein A- Sepharose column (Pharmacia) and from ascites by passage through Blue Sepharose and anion exchange chromatography. Antibodies from these cell lines were used in the Western Blot analysis of the cyanogen bromide fragments of OSN p40, discussed below. Preparation of PBL

Venous blood from patients with cancer or unrelated diseases was drawn into heparinized Vacutainer tubes, buffy coat leukocytes (PBL) were isolated, and red blood cells (RBC) were lysed, as described previously.

7 In all experiments, 1 x 10 PBL were first incubated with 2.5 x 10^~6M PGE, in 0.5 ml Medium 199 for 5 min at 20° and then diluted to 1 x 10 7 cells/ml in medium 199.

PBL were preincubated with PGE, because leukocytes from patients with large tumor burdens or after surgery tend to be nonadherent and do not react in the LAI assay.

PGE_ restores the adherence of leukocytes to that of normal subjects. Leukocytes from control subjects were treated similarly. Tube LAI Assay

Cancer extract-induced LAI: The tube LAI assay was performed in 20 ml, 16 x 150mm glass test tubes in triplicate, as previously described in Grosser and Thomson (1075) Cancer Res. 35:2571-2579. Medium T99 (0.3 ml), 0.1 ml cancer extract such as lung (=100 yg protein), and 0.1 ml suspended leukocytes were added to three tubes. To another three tubes, 0.3 ml medium

199, 0.1 ml of another cancer extract such as pancreas, and 0.1 ml suspended leukocytes were added. The tubes were laid flat and incubated at 37°C in a 5% C0_ humidi¬ fied atmosphere. Two hours later, the tubes were placed upright, the contents at the bottom were gently agitated with a Pasteur pipette, and a sample of the nonadherent cells in each tube was placed onto a hemacytometer and counted by computer-driven image analysis. The computer- linked instruments calculated the mean number of non¬ adherent cells and expressed the results in terms of the percentage change in the nonadherence index (NAI): NAI = [ (A - B)/B] x 100, where A equals the number of nonadherent cells in the sample after incubation with the first cancer extract and B equals the number of nonadherent cells in the sample after incubation with the control cancer extract. In previous studies, leuko¬ cytes from more than 95% of the subjects without cancer had NAI less than 30, distributed normally about zero, whereas leukocytes from most patients with early cancer similar to that in tube A had NAI greater than 30.

Peptide induced LAI: The ability of the pep¬ tides of p40 lung cancer OSN to inhibit the adherence of leukocytes to glass was compared to that of p40 lung cancer OSN with a modification of the above assay. To three tubes, 0.3 ml medium 199, 0.1 ml (=100 yg) control g pancreatic cancer extract, 0.1 ml of 1 x 10 suspended leukocytes, and 0.01 ml p40 or a peptide appropriately diluted in medium 199 were added. To another three tubes, 0.3 ml medium 199, 0.1 ml (-100 yg) of the lung cancer extract, and 0.1 ml suspended leukocytes were added. After 2 hours of incubation, the nonadherent cells were counted by image analysis and the results calculatd as above; except that A now equals the number of nonadherent cells found in a sample after incubation with p40 or its peptide and B equals the number of non¬ adherent cells found in a sample after incubation without p40 or its peptide. In other experiements, 100 yl of FBS previously diluted to 1% in medium 199 was substituted for the control cancer extracts with the same results. High-dose inhibition (blocking) LAI: The assay was conducted as previously described. In this assay, the conditions were set up identical to that described above with p40 or peptide added to tubes A at a concentration giving a peak response (0.75 yg/tube) . The effect on LAI of the addition of another substance to tube A was then evaluated. Medium alone, intact p40, cleaved or digested p40, and the peptides of p40 were added in 0.01 ml to tubes A. The assays were per¬ formed and read as described above. The peak response to p40 was compared to the peak response of p40 plus the additions. Peptide Synthesis

Peptides were synthesized using the Merrifield solid-phase methodology on 4-methylbenzhydrylamine resin.

The product was cleaved from the resin and the protecting groups were removed using anhydrous liquid hydrogen fluoride. Each peptide was synthesized both as the unmodified compound (C-terminal carboxamide and N-terminal acetate) and with the three amino acid appendage (cysteine- glycine- glycine) at the N-terminal. The appended peptide was coupled to keyhole limpet hemocyanin through its N-terminal cysteine via either maleimido-benzoyl-N- hydroxysuccinimide (MBS) or N-succinimidyl bro oacetate.

The coupled peptides were used for immunizing mice and rabbits via standard procedures.

RESULTS

Reversed-phase HPLC of Lung p40 Peptides, and Preparation and Testing of Synthetic Peptides The reversed-phase HPLC peptide maps of lung p40 resulting from various of the cleavage methods are shown in Figs. 1-6. Each method produces a different set of peptide fragments depending on its specificity. All of the methods produce large fragments (except the chymotrypsin cleavage) due to the nature of the residue at which they cleave. For example, cyanogen bromide cleaves at methionine, limited acid hydrolysis cleaves at aspartic acid-proline bonds, and chymotrypsin cleaves mainly at aromatic residues. Anhydrous HF cleaves the carbohydrate moieties from the protein while leaving the peptide backbone intact. By looking at the amino acid composition, the number of fragments expected from each cleavage method can be estimated.

The peptide fragments from the cyanogen bromide (CNBr) cleavage were characterized in detail. The major fragments were analyzed for their amino acid composition and amino acid sequence. These analyses were performed at the Protein Analysis Laboratory, Scripps Clinic and Research Foundation, La Jolla, California. Table 1 lists the amino acid composition of lung CM p40 and its seven major CNBr fragments. Approximately 30 peaks were observed in the HPLC peptide map. Table 2 sets forth the sequence analysis of five .of these cyanogen bromide (CN) fragments as well as four additional chymotryptic (CH) fragments.

TABLE 1: Amino Acid Composition of Cyanogen Bromide Fragments*

Res CM p40 CN-2 CN-4 CN-5 CN-6 CN-8 CN-10 CN-12

ASP 48 23.9 (24) 9.2 ( 9) 12.5 (13) 10.9 (11) 7.9 ( 8) 42.7 (43) 34.5 (35)

THR 22 11.8 (12) 3.9 ( 4) 5.3 ( 5) 4.7 ( 5) 2.3 ( 2) 15.8 (16) 9.8 (10)

SER 18 11.4 (11) 5.4 ( 5) 6.2 ( 6) 4.0 ( 4) 1.0 ( 1) 9.2 ( 9) 7.2 ( 7)

GLU 40 14.8 (15) 6.5 ( 7) 7.7 ( 8) 8.9 ( 9) 7.7 ( 8) 19.9 (20) 9.4 ( 9)

PRO 11 3.5 ( 4) 1.4 ( 1) 1.3 ( 1) 2.5 ( 3) 2.4 ( 2) 7.5 ( 8) 7.2 ( 7)

GLY 21 7.4 ( 7) 5.1 ( 5) 4.2 ( 4) 4.9 ( 5) 3.3 ( 3) 11.6 (12) 10.9 (11)

ALA 18 11.0 (11) 4.6 ( 5) 4.5 ( 5) 5.0 ( 5) 2.4 ( 2) 8.5 ( 9) 9.5 (10)

VAL 18 1.1 ( 1) 1.0 ( 1) 0 3.7 ( 4) 4.8 ( 5) 15.8 (16) 7.1 ( 7)

MET 2

ILE 16 4.9 ( 5) 2.6 ( 3) 3.1 ( 3) 4.3 ( 4) 4.4 ( 4) 9.7 (10) 4.4 ( 4)

LEU 24 8.8 ( 9) 4.4 ( 4) 5.6 ( 6) 5.4 ( 5) 4.6 ( 5) 16.9 (17) 12.7 (13)

TYR 10 2.1 ( 2) 1.3 ( 1) 1.0 ( 1) 2.9 ( 3) 3.6 ( 4) 4.5 ( 5) 2.8 ( 3)

PHE 23 11.5 (12) 4.8 ( 5) 6.4 ( 6) 5.0 ( 5) 2.7 ( 3) 17.2 (17) 13.9 (14)

HIS 6 5.0 ( 5) 2.0 ( 2) 2.6 ( 3) 1.5 ( 2) 0 1.0 ( 1) 1.0 ( 1)

LYS 41 20.0 (20) 6.8 ( 7) 9.3 ( 9) 10.1 (10) 6.1 ( 6) 28.4 (28) 21.1 (21)

ARG 5 5.4 ( 5) 1.5 ( 2) 2.3 ( 2) 1.0 ( 1) 0 0 0

TRP 5

CYS 2

00 * Normalized molar ratios. 00 o

Table 2

CN-2: (N) NTFNNETILH GKDAXQ (C)

CN-6: (N) GTFLNFX-_^ILN GKXX₂ (C)

CN-8: (N) EALIVTNPIP YDVGVFRKSV

NQLEQNLIVQ TFINLAKNKQ DTYGPLTGYN GYKXI (C)

CN-10: (N) LNKKKGGKEV NKEV (C)

CN-12: (N) LSKKKGGKEV NKEVTNTFLE

NFLXEN (C)

CH-13: (N) NNEANEFMEA L (C)

CH-18: (N) AAWK (C)

CH-22: '(N) INENAKDN (C)

CH-29: (N) IVTNPIPY (C)

x = unknown X_χ = T or G X₂ = N or A

The CNBr fragments of lung p40 were analyzed by SDS-PAGE and then transferred to nitrocellulose and probed with a panel of antibodies to the native protein. These results are summarized in Table 3. The SDS-PAGE results yields the molecular weight of the fragment (and purity) and the Western-Blot analysis indicates the presence of antigenic sites in the fragments which are in the native protein. Western Blot analysis showed the CN-10 and CN-12 fragments were recognized by two of three monoclonal antibodies (A, B, and C) raised against the intact lung OSN p40.

Table 3 CNBr Major Antibody Reactivity

Peptide Mr Gel Bands A B C

CN-2 13

11

CN-5 13 11

CN-6 28 X

24 X

22 X

9

CN-8 X

+ +

+ + + +

+

+ + + + + +

The p40 cyanogen bromide peptide fragments were resolved on 16% acrylamide SDS gels, then blotted onto nitrocellulose and immunoreacted with the three monoclonal antibodies. LAI Response to Intact, CNBr Cleaved, HF Cleaved, and Tryptic Digested Lung p40 OSN

Leukocytes from patients with lung cancer were tested to different concentrations of intact p40 OSN. The dose-response curve was narrow and bell-shaped (See Fig. 7). Values in Fig. 7 are arithmetic mean of at least five different subjects tested in duplicate. Zero antigen concentration indicates the NAI values to the same particulate extracts in tubes A and B without p40 OSN. Values >30 are significantly different from zero values (p<0.001). The response of the same leukocytes to lung cancer extract in tube A and pancreatic cancer in tube B is shown on the right. The peak response was from 0.5 to 0.75 yg p40 per test tube. Leukocytes from subjects without cancer or with malignant melanoma had no response to the same concentrations as well as to both higher and lower concentrations than those shown in Fig. 7. CNBr, HF cleaved, or tryptic digested p40 gave a similar dose-response curve and peak response (Tables 4, 5, and 6). The results indicated that that antigenic epitope(s) of p40 were not destroyed by CNBr, tryptic digestion, or HF cleavage and that the epitope(s) must be linear determinant(s) that were recognized by class-II restricted T helper cells. TABLE 4: Dose Response in LAI of Leukocytes to CNBr Cleaved p40 Lung Cancer OSN

Leukocyte NAI to different concentrations of CNBr cleaved p40 Donors 0.25 0.5 0.6 0.75 1.0 (yg)

Lung Cancer 8+6 42+10 39+13 61+11 14±8

Malignant melanoma 2±4 0±5 7±11 3±8 5+3

Control subjects 6±3 ^•9±5 •5±6 7±10 4±4

TABLE 5: Dose Response in LAI of Leukocytes to HF Cleaved p40 Lung Cancer OSN

Leukocyte NAI to different concentrations of HF cleaved p40 Donors 0.1 0.25 0.5 0.75 1.0 (yg)

Lung Cancer 3±4 22±11 56+8 66+10 -4±2

Malignant melanoma ^■2±3 -3±7 0+5 1+4

Control subjects 3±5 2±5 0+8 •2±6

TABLE 6: Dose Response in LAI of Leukocytes to Tryptic Digest of p40

Leukocyte NAI to different concentrations Donors 0.1 0.25 0.5 0.75 1.0 1.25 (yg)

Lung Cancer 8.0+11 7.0±10 85±17 74±18 34±15 -3±7

Malignant melanoma 3±5 0+9 6±5 2±4 1±8

Control subjects 11±4 -2±8 10±13 -6±3 -3±7

The CNBr fragments of p40 lung cancer were separated by reversed-phase HPLC, as described above. The individual peaks were tested by high-dose inhibition. Two different preparations were prepared, isolated, and tested. In the^" first preparation, peak eight gave high dose inhibition, and when tested as antigen it gave a positive peak response at about 0.2 to 0.25 yg. In the second preparation, peak 15 gave high dose inhibition, and when tested as antigen gave a positive peak response at 0.25 yg. Peaks 8 and 15 correspond to peptide CN-8 described in Tables 1, 2, and 3, above.

Based on these results, the peptide of peak 8 (CN-8) of the first preparation was sequenced, and two synthetic peptides corresponding to sequences within CN-8 were prepared, as described above. The synthetic sequences were as follows:

Peptide 1: FINLAKNKQD TYGPL

Peptide 2: TNPIPYDVGV FRKSVNQL Both peptides were also prepared with a cysteine-glycine- glycine sequence appended to the amino terminal end. Such appendage was used to conjugate the peptide to keyhole limpet hemocyanin to form an immunogen useful in the preparation of antibodies, as described above. The synthetic peptide designated peptide 1 was extensively tested in the LAI assay. Table 7 shows dose-response testing to synthetic peptide 1 by leukocytes from lung or breast cancer patients and control subjects. Leukocytes from lung cancer patients showed positive peak responses at 0.175 to 0.225 yg of peptide 1. Leuko¬ cyte from control subjects or breast cancer patients did not react. The Table 8 shows the response of dif¬ ferent cancer patients to LAI testing with peptide 1. Positive responses were found only with leukocytes from lung cancer patients. Although a few patients with breast cancer had higher responses than controls, the response was significantly different from that of lung cancer patients.

TABLE 7: Testing by Dose-response of Synthetic Peptide 1 in LAI Assay

Leukocyte NAI to different concentrations (yg of Peptide 1) Donors 0.05 0.1 0.15 0.175 0.2 0.225 0.25 0.3

Corresponding to Lung p40 CNBr Digest Fragment B Peptide 1

*

0.2 yg of peptide added to tube A.

To verify the specificity of peptide 1, coded samples were also tested. Tables 9 to 14 show the results, Tryptic digest of p40 lung cancer or colon cancer were recognized specifically (Table 9). Tables 10 to 14 show that peptide 1, whether LAI tested as an antigen or by high-dose inhibition, triggered responses with leukocytes only from lung cancer patients.

* TABLE 9: Coded Testing by LAI of Tryptic Digest of

Pure p40 from Colon Cancer and Lung Cancer

Patient Diagnosis NAI to Samples A B

Colon cancer (N=3) 5±7 35±9 Lung cancer (N=3) 35±2 15±7 <0.01 <0.05

Code: A=p40 lung cancer; B=p40 colon cancer.

TABLE 10: Coded and Fixed Concentration Testing by LAI of Synthetic Lung Cancer Peptide (#i), Control Protein and Synthetic Nonapeptide T18 of Myelin Basic Protein (MBP)

Patient Diagnosis NAI to Sam les

Colon cancer (N=2) Lung cancer (N=2) Significance

Code: A = T18 peptide; B = control protein; C = synthetic lung peptide.

NS= Not significant. *

TABLE 11: Coded Testing by High-dose Inhibition of LAI with Synthetic Lung Cancer Peptide 1, Control Protein, and Synthetic Nonapeptide T18 of MBP

NAI to NAI (% inhibition) to samples

Patient Crude Diagnosis Cancer Extract

Colon Cancer 33+5 -1±31 35±10 32±8

(N=2) (103) (-3) (3)

Significance <0.01 NS NS

Lung Cancer 40±8

(N=2)

Significance

*

Code: A = T18 peptide; B = control protein; C = synthetic lung peptide. NS = not significant.

^•A*

TABLE 12: Coded and Dose-response Testing by LAI with Synthetic Lung Peptide 1 and Synthetic T18 of MBP

NAI to Samples A

Patient Diagnosis 0. 15 0. 175 (yg) 0. 125 0. 15 (yg)

Colon Cancer (N=5) -3±15 15±6 33±9 42±12 Lung Cancer (N=5) 40±9 51±10 32+7 6L+14 Significance <0.001 <0.01 NS NS

* Code: A = synthetic lung peptide; B = synthetic T18 of MBP. NS= not significant. *

TABLE 13: Coded Testing by High-dose Inhibition of LAI with

Synthetic Lung Cancer Peptide 1 and Tryptic Peptide C D of Colon Cancer Purified by RP-HPLC

NAI to NAI (% inhibition) to Samples Patient Crude Cancer

.Diagnosis Extract

0.3 0.4 (yg) 0.3 0.4 (yg)

-11+4 -2±6 A N S

(122) ^■ (104) B <0.001

10

38±7 45±20 A <0.001 (34) (22) B N S

, i- Signif cance <.001 <0.05 <0.005 <0.05

*

Code: A = lung synthetic peptide 1; b = tryptic peptide of p40 colon (13 residues).

0

TABLE 14: Coded and Dose-response Testing by LAI with Synthetic L Luunngg CCaanncceerr PPeeppttiiddee 11 aanndd Tryptic Peptide C.D of Colon Cancer Purified by RP-HPLC

5 NAI to Samples

Patient A (μl) B (μl) Diagnosis 9 10 9 10

Colon Cancer (N=3) 53±8 44±8 -12±3 -14±5 0 Lung Cancer (N=3) -5+4 -10+8 47±21 39±11 Significance <0.001 <0.005 <0.05 <0.005

rt

Code: A = colon tryptic peptide of p40 (13 residues); B = lung cancer synthetic peptide. 5 In conclusion, synthetic peptide 1 is a deter¬ minant of the p40 OSN which is recognized by leukocytes from lung cancer patients but is not recognized by leuko¬ cytes from patients with other cancers. Colon p40 Peptides

Reversed-phase HPLC maps of colon CM p40 result¬ ing from CNBr and tryptic cleavage methods are shown in Figs. 8 and 9, respectively. Major peaks from both digests were sequenced, and the sequence analyses are set forth in Table 15.

Table 15 1. Cyanogen Bromide Fragments

Code Sequence CCN-10 (N) E-A-L-I-V-T-N-P-I-P-Y-D-V-G-V-F-R-K-S-

V-N-Q-L-E-Q-N-L-I-V-Q-T-F-I-N-L-A K-N-K-Q -D-T-Y-G-P-L (C) CCN-15 (N) L-N-K-K-K-G-G-K-E-V-N-K-E-V-V-N-

T-F-L-E-N-X-K (C) CCN-13 (N) L-N-K-K-K-G -G-K-E-V-N-K-E-V-S-N-T-

X-X₃-E-N-X-K-G-G (C) CCN-8 (N) N-T-F-L-N-V-T-X₄-L-X₅-G (C)

Tryptic Fragments

Code Sequence

Q-S-V-Y-D-P-T-V-Q-A-N (C) V-S-Q-G-Q-W-D-K (C) F-Y-V-D-G-T-D-S-D-P-L-V-K (C) Y-V-D-G-T-D-S-D-P-L-V-K (C)

W-N-G-N-V-T (C)

A-S-W-T-D-E-N-H-K (C) I-D-N-F-Q-K (C)

F-T-T-L-N-E-D-R (C) I-F-V-E-T-P-Y (C)

d X_ς defined hereinabove.

The tryptic and cyanogen bromide fragments of the colon p40 OSN were characterized in the same manner as the corresponding lung antigen fragments. Both sequence data and LAI activity were obtained for the major fragments. The most interesting fragments were then prepared by solid-phase synthesis. In the case of the cyanogen bromide cleavage, all of the major peaks of the reversed-phase HPLC map were screened in the LAI assay by high-dose inhibition. Table 16 indicates the results of these assays and the six peaks which were considered positive. The peak ^"numbering corresponds to the numbering in the reversed phase HPLC trace (Fig. 8).

The individual active peaks from the digest were retested to verify their activity. For example, Table 17 shows the coded testing of the major active fragment (#10) from the cyanogen bromide cleavage.

The tryptic fragments were treated analogously. The peaks from the reversed-phase HPLC peptide map (Fig. 9) were screened in the LAI assay by high-dose inhibition and the active peaks were indicated (Table 18). The positive fractions were then screened a second time as antigen in the LAI assay to verify their activity (Table 19). All active fractions were sequenced as described above (Table 15). Coded testing by high-dose inhibition and dose-response testing of a major active tryptic fragment (C..D) is shown in Tables 13 and 14.

TABLE 16: Results of Screening in LAI by High-Dose

Inhibition of Colon p40 OSN CNBr Cleavage Products Isolated by RP-HPLC

+

+

The HPLC sample fractions correspond to the numbers of the peaks in the colon antigen cyanogen bromide digest peptide map (Fig. 8).

TABLE 17: Coded testing by High-Dose Inhibition of LAI with Synthetic Lung Cancer Peptide 1 and CNBr Cleavage Product of p40 Colon Cancer Isolated by RP-HPLC.

Diagnosis of NAI to NAI (% Inhibition) to Samples

* Leukocyte Donor Crude Extract A B Lung Cancer 59 ± 8 (0) 12+8 ( 103)

(n = 4) 59 + 13 Colon Cancer 4 + 4 (96) 5L+11 (39 )

(n = 4) 83 i 22 p<0. 005 p<0. 005

A = Colon p40 CNBr cleavage separated on C18 RP-HPLC 10 from

Bio-Rad.

B Synthetic lung peptide 1 n Number tested

Crude cancer extract used is identified with diagnosis of donor leukocytes

TABLE 18: Screening by High-Dose Inhibition of Tryptic Digest of Colon Cancer p40 Isolated on C RP-HPLC

Peaks marked with asterisks (*) have been sequenced. RP-HPLC peak labels correspond to tryptic digest peptide map (C-3 column). TABLE 19: Repeat Screening as Antigen in LAI from C3 RP-HPLC of

Tryptic Digest Colon Cancer p40 of Positive Fractions as Determined by High-Dose Inhibition Franction from NAI

C3 RP-HPLC 250 (μg) 200 (μg)

Bl A 75 42

C 30 41

D 30 13

18 -14

The tryptic fragment C..D (Table 15) was prepared synthetically, as described above, in two forms.

Peptide 3: (N) FYVDGTDSDP LVK (C) Peptide 3: (N) CGGFYVDGTD SDPLVK (C) (Appended)

The appended form was conjugated to keyhole limpet hemocyanin and used to prepare antibodies, as described above.

Dose-response LAI testing of synthetic peptide 3 is shown in tables 20 and 21. The result of these experiments is that the peptide is a determinant of the colon p40 OSN which is recognized by leukocytes from colon cancer patients and not by leukocytes from lung cancer patients. TABLE 20 : Dose Response Testing in LAI of Synthetic Peptide of

Colon Cancer peptide 3. Diagnosis of NAI to NAI to Different Concentrations

Leukocyte Donor Crude Extract* of Synthetic Peptide (μg)

0. 15 0. 175 1. 2 0. 225 0. 25

Colon Cancer 52 ± 5 1±9 15±28 58±18 87±21 31±9

Lung Cancer 00 ± ι5 -6±8 - 1±7 5+12 -4±8 1±6

* Crude extract similar to donor leukocytes

TABLE 21 : Coded Study by Dose-Response Testing by LAI of Synthetic Lung Cancer Peptide 1, Synthetic Colon Peptide 3 and a Nonsense Peptide.

NAI to Sam les Diagnosis of Leukocyte Donors Lung Cancer (n=3)

A = Nonsense peptide B = Synthetic colon peptide 3 C = Synthetic lung peptide 1.

Antibodies against peptides 1 , 2 , and 3 , were prepared by the methods described above . Various anti sera against each of the peptides were then tested against the peptides , as well as against both lung and colon p40 , by enzyme-linked immunoabsorbent assay (ELISA) , Western blot analysis , and radioimmunoassay ( RIA) . The results are set forth in Table 22 .

Table 22

Reactivity with: Antisera Pept . 1 Pept. 2 Anti-peptide 1 No. 1

2

3 ++

4

5

6 +/•

Anti-peptide 2 No. 1 2 3 4 5 6 ■1

Anti-peptide 3 No. 1 + ++ 2 + + 3 + +

+++ = very strongly positive ++ = strongly positive + = positive +/- = weakly positive - = negative ND = not done

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims,

Claims (21)

WHAT IS CLAIMED IS:

1. A peptide fragment having no more than 100 amino acids and being immunologically cross-reactive with an organ-specific neoantigen (OSN) capable of inducing leukocyte adherence inhibition (LAI) in the leukocytes of a patient having a solid tumor.

2. A peptide fragment as in claim 1, which is cross-reactive with neoantigen from a particular type of tumor, but substantially free from cross-react¬ ivity with neoantigen from all other types of tumor.

3. A peptide fragment as in claim 2, which is cross-reactive with lung neoantigen.

4. A peptide fragment as in claim 1, which is prepared by proteolytic cleavage of the organ-specific neoantigen.

5. A peptide fragment as in claim 4, which is prepared by cyanogen bromide cleavage of the organ- specific neoantigen.

6. A peptide fragment as in claim 1, which is prepared by chemical synthesis.

7. A peptide fragment as in claim 1 which is prepared by expression of a recombinant vector in a cell culture host.

8. A peptide as in claim 1 which peptide is cross-reactive with the epitope on the OSN which causes LAI.

9. A peptide ragment having at least 80% homology with one of the following amino acid ^"sequences or a portion thereof:

(I) GTFLNFX-_^ILN GKXX₂;

(II) EALIVTNPIP YDVGVFRKSV NQLEQN IVQ TFINLAKNKQ DTYGPLTGYN GYKXI;

(HI) LNKKKGGKEV NKEV;

(IV) LSKKKGGKEV NKEVTNTFLE NFLXEN.

(V) LNKKKGGKEV NKEWNTFLE NXK

(VI) NNEANEFMEA L

(VII) AAWK

(VIII) INENAKDN

(IX) IVTNPIPY

(X) LNKKKGGKEV NKEVSNTXX₃E

NXKGG

(XI) NTFNNETILH GKDAXQ

(XII) NTFLNVTX₄LX₅

(XIII) QSVYDPTVQA N

(XIV) VSQGQWDK

(XV) FYVDGTDSDP LVK (XVI) YVDGTDSDPL VK

(XVII) WNGNVT

(XVIII) ASWTDENHK

(XIX) IDNFQK

(XX) FTTLNEDR

(XXI) IFVETPY.

10. Antibodies raised against the peptides of claim 1

11. Antibodies raised against the peptides of claim 9.

12. A method for detecting the presence of an organ-specific neoantigen in a biological specimen, said method comprising: exposing the specimen to an antibody raised against a peptide fragment having no more than 100 amino acids and being immunologically cross-reactive with an organ-specific neoantigen (OSN) capable of inducing leukocyte adherence inhibition (LAI) in the leukocytes of a patient having a solid tumor.

13. A method as in claim 12, wherein the peptide fragment is cross-reactive with neoantigen from a particular type of tumor, but substantially free from cross-reactivity with neoantigen from all other tumor types.

14. A method as in claim 12, wherein the peptide fragment is cross-reactive with neoantigen from at least two types of tumor.

15. A method as in claim 12, wherein the patient sample is a serum sample.

16. A method for detecting the presence of an organ-specific neoantigen in a patient blood sample, -said method comprising: exposing leukocytes from the blood sample to OSN peptides; and observing inhibition of leukocyte binding as a result of the exposure to OSN.

17. A method as in claim 15, wherein the OSN peptides are a mixture of peptides corresponding to different determinant sites on an OSN p40.

18. A method as in claim 15, wherein the OSN peptides are purified to at least about 80% w/w and corresponds to a single determinant site on OSN p40.

19. A method as in claim 15, wherein the OSN peptides correspond to lung cancer OSN.

20. A method as in claim 15, wherein the OSN peptides correspond to colorectal cancer OSN.

21. A method as in claim 16, wherein the OSN peptides correspond to a determinant site common to OSN's from at least two types of tumor.