EP0407451A1 - Facteur general de detection de scm lie au cancer; preparation et procede d'utilisation - Google Patents

Facteur general de detection de scm lie au cancer; preparation et procede d'utilisation

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Publication number
EP0407451A1
EP0407451A1 EP19890904468 EP89904468A EP0407451A1 EP 0407451 A1 EP0407451 A1 EP 0407451A1 EP 19890904468 EP19890904468 EP 19890904468 EP 89904468 A EP89904468 A EP 89904468A EP 0407451 A1 EP0407451 A1 EP 0407451A1
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EP
European Patent Office
Prior art keywords
scm
factor
phe
lymphocytes
cancer
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EP19890904468
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German (de)
English (en)
Inventor
Boris Cercek
Lea Cercek
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Individual
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4745—Cancer-associated SCM-recognition factor, CRISPP
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • cancer in the treatment of the various malignancies that afflict humans and animals, referred to generally as cancer, it is recognized that early detection is the key to effective treatment, especially as most therapeutic procedures are more effective and safer in relatively early stages of cancer than in later stages.
  • many chemotherapeutic drugs that are toxic to malignant cells are also toxic to normal cells, and the higher doses required to cure or arrest more advanced cases of cancer can cause uncomfortable and serious side effects.
  • surgery is most often effective only before the disease has spread or metastasized. Far too many cases of cancer are only discovered too late for effective treatment.
  • test ability either to detect all types of cancer generally, or to detect specific types of cancer, depending on the materials used.
  • the former application of such a test is very important in mass screenings of large patient populations, as would be done in routine checkups.
  • mass screenings a test dependent on a particular type of cancer would not be desirable, as there are literally hundreds, if not thousands, of types of cancer and a test that could spot only one or a few types of the disease is far too likely to miss many cases of cancer.
  • these patients would present either no symptoms or vague generalized symptoms that could not be readily linked to a particular type of cancer, so there would be no basis for suspecting a particular type and administering a test specific for that type.
  • a subpopulation of potentially SCM-responding lymphocytes is separated from a blood sample of the patient being tested and the lymphocytes are incubated with malignant tissue or extracts of malignant tissue. If the blood sample donor is afflicted with a malignancy, there will be a characteristic SCM response that can be differentiated from the SCM response of lymphocytes from donors not afflicted with a malignancy.
  • the SCM response is determined by measuring changes in intracellular fluorescein fluorescence polarization of the SCM-responding lymphocytes. The changes seen in the SCM test are believed to reflect changes in the internal structure of the lymphocyte as the lymphocyte is activated for synthesis.
  • Fluorescence polarization is a measure of intracellular rigidity; the greater the intracellular mobility, the less the measured fluorescence polarization.
  • An observed decrease in fluorescence polarization is thought to result mainly from changes in the conformation of the mitochondria, the energy-producing organelles of the cell. The change in the mitochondria is believed to result from the contractions of the cristae or inner folds of the mitochondrial membrane.
  • the SCM reflects the forces of interaction between macromolecules and small molecules such as water molecules, ions, adenosine triphosphate, and cyclic adenosine phosphate. Perturbations of these interactions result in changes in the SCM.
  • the SCM test is capable of responding to a relatively small quantity of malignant cells. About 10 9 cells in a person weighing 70 kg are enough to cause the lymphocytes to respond in the SCM test in the characteristic pattern of malignancy. In mice, when as few as 7.5 x 10 5 Ehrlich ascites (tumor) cells are implanted, the pattern of the response in the SCM test is altered; response to cancer-specific antigens is induced, while the normal response to phytohaemagglutinin is virtually eliminated (L. Cercek and B. Cercek, "Changes in SCM-Responses of Lymphocytes in Mice After Implantation with Ehrlich Ascites Cells," Europ. J. Cancer 17, 167-171 (1981)).
  • the SCM test allows early detection of cancer, often much earlier than is possible by conventional methods , with relatively little discomfort to the patient except as may be involved in taking a blood sample.
  • this procedure does have disadvantages. For example, it requires preparation of crude extracts from tumor tissues and the like or the use of the tumor tissue itself as a source of cancerassociated antigens.
  • the use of whole tissues or crude extracts of tissues can introduce interfering substances into the test procedure. These interfering substances can adversely affect the sensitivity of the test or adversely affect the test results themselves. The presence or absence of these interfering substances can easily vary from batch to batch of malignant tissue, introducing undesirable variability into the SCM test. Additionally, because the interfering substances are present in whole tissue or crude extracts, they are very difficult to identify or quantitate.
  • cancer recognition factors in animal and human body fluids, more particularly in blood plasma and urine, which, if the donor body is afflicted with cancer, produce a response in SCM-responding lymphocytes that is identical to the response by such lymphocytes to cancer-associated extracts and/or tumor tissue in the SCM test.
  • the factors are designated in the singular and referred to as the "general cancer-associated SCM-recognition factor,” the “SCM-recognition factor,” or merely as the "SCM factor.”
  • the SCM factor is believed to be a peptide or peptide-like material and to comprise several closely related peptides.
  • the SCM factor is believed to include a thirteen-amino-acid fragment having the sequence Phe-R 1 -Lys-Pro-Phe-R 2 -Phe-R 3 -Met-R 4 -R 5 -R 6 -R 7 , wherein R 1 is selected from the group consisting of Asn and Gln, R 2 , R 3 , and R 4 are each independently selected from the group consisting of Val, Leu, and Ile, R 5 is selected from the group consisting of Asp and Glu, and R 6 and R 7 are each independently selected from the group consisting of Asn and Gln. This sequence does not include the amino terminus of the SCM factor.
  • the general cancer-associated SCM-recognition factor consists essentially of low molecular weight peptide passing through filters with a nominal 1000- dalton molecular weight cutoff and retained by filters with a nominal 500-dalton molecular weight cutoff, and producing at least a ten percent decrease in the intracellular fluorescence polarization value of SCM- responding lymphocytes isolated from donors afflicted with cancer when used to challenge the lymphocytes in the standard SCM test.
  • the factor purified by the process described below has the approximate amino acid composition of (Asx 2 , GIX 3 , Ser, His, Gly 5 , Thr, Arg, Ala 3 , Tyr, Met, Val 3 , Phe 3 , Ile, Leu 3 , Lys 2 ).
  • a sixteen-aminoacid segment within one preparation of the factor has the amino acid sequence Phe-Asn-Lys-Pro-Phe-Val-Phe-Leu-Met-Ile-Asp-Gln-Asn-Phe-Ser-Lys, the segment not including the amino terminus of the factor.
  • a fifteen-amino-acid segment within another preparation of the factor has the amino acid sequence Phe-Asn-Lys-Pro-Phe-Val-Phe-Leu-Met-Ile-Asp-Gln-Asn-Thr-Lys, the segment also not including the amino terminus of the factor.
  • an important additional aspect of the invention is an SCM factor comprising only peptides of defined sequence, the peptides either having been shown directly to have SCM activity or believed to have such activity.
  • such a factor comprises substantially only peptides having at least thirteen amino acid residues including a sequence of Phe-R 1 -Lys-Pro-Phe-R 2 -Phe-R 3 -Met-R 4 -R 5 -R 6 -R 7 therein, where R 1 is selected from the group consisting of Asn and Gln, R 2 , R 3 , and R 4 are each independently selected from the group consisting of Val, Leu, and Ile, R 5 is selected from the group consisting of Asp and Glu, and R 6 and R 7 are each independently selected from the group consisting of Asn and Gln.
  • this factor contains substantially only one peptide.
  • the first class most broadly comprises substantially only peptides having at least fifteen amino acid residues including a sequence of Phe-R-(-Lys-Pro-Phe-R 2 -Phe-R 3 -Met-R 4 -R 5 -R 6 -R 7 -R 8 -Lys therein, wherein R 1 through R 7 are as defined above and R 8 is selected from the group consisting of Ser and Thr.
  • this factor contains substantially only one peptide.
  • a more closely defined factor comprises substantially only peptides including a sequence of Phe-Asn-Lys-Pro-Phe-Val-Phe-Leu-Met-Ile-Asp-Gln-Asn-Thr-Lys therein. These peptides can include additional flanking sequences on either side of the specified sequence. Preferably this factor contains substantially only one peptide.
  • the most closely defined factor within this class comprises a substantially pure peptide consisting essentially of the amino acid sequence Phe-Asn-Lys-Pro- Phe-Val-Phe-Leu-Met-Ile-Asp-Gln-Asn-Thr-Lys.
  • the second class most broadly comprises substantially only peptides having at least sixteen amino acid residues including a sequence of Phe-R 1 -Lys- Pro-Phe-R 2 -Phe-R 3 -Met- R4-R5-R6 -R 7 -Phe-R 8 -Lys therein, wherein R 1 through R 7 are as defined above and R 8 is selected from the group consisting of Ser and Thr .
  • this factor contains substantially only one peptide .
  • a more closely defined factor comprises substantially only peptides including a sequence of Phe-Asn-Lys-Pro-Phe-Val-Phe-Leu-Met-Ile-AspGln-Asn-Phe-Thr-Lys therein. These peptides can include additional flanking sequences on either side of the specified sequence. Preferably this factor contains substantially only one peptide.
  • the most closely defined factor within this class comprises a substantially pure peptide consisting essentially of the amino acid sequence Phe-Asn-Lys-ProPhe-Val-Phe-Leu-Met-Ile-Asp-Gln-Asn-Phe-Thr-Lys.
  • this peptide has an activity in the SCM test such that 5 x 10 -2 femtograms of the peptide produce a decrease in polarization value of at least 30 percent when used to challenge SCM-responding lymphocytes from donors afflicted with cancer.
  • the SCM factor can be produced by ultrafiltering a body fluid from a donor afflicted with cancer in order to separate a first fraction of the body fluid comprising molecules having an apparent molecular weight greater than 1000 daltons from a second fraction comprising molecules having an apparent molecular weight of less than 1000 daltons.
  • the body fluid is selected from the group consisting of peripheral blood, urine, and plasma.
  • the factor undergoes a further purification process comprising several stages, each stage resulting in a more highly purified factor.
  • the first stage of this purification process comprises elution from a gel filtration column with a fractionation range of from 0 to about 700 daltons and capable of separating the salts from the ultrafiltrate, the factor eluting at between about 0.3 and about 0.5 times the total chromatographic bed volume.
  • the second stage comprises elution from a gel filtration column having a fractionation range of from about 1500 daltons to about 30,000 daltons, the factor eluting from such a column at between about 0.4 and about 0.6 times the total chromatographic bed volume.
  • the next stage of this purification process comprises elution from an anion-exchange column of diethylaminoethyl cellulose at between about 0.28 M to about 0.31 M of ammonium bicarbonate.
  • the final stage comprises purifying the factor to substantial homogeneity by reverse-phase highpressure liquid chromatography.
  • the factor from any stage of the purification including the initial ultrafiltrate, can be used in the test.
  • the SCM factor produces a positive response in potentially SCM-responding lymphocytes derived from donors having a variety of different types of malignancies, regardless of the type of cancer present in the donor from whom the SCM factor is isolated; hence the term "general.”
  • the factor provokes little or no SCM response in lymphocytes derived from donors free of malignancies. This ability to act as a general cancerassociated antigen makes the factor useful for the general screening of blood samples for the presence of malignancy.
  • the factor also has other properties of interest.
  • One of these properties is the property of modifying the SCM response of SCM-responding lymphocytes obtained from donors free of malignancy when the factor is contacted with such lymphocytes, whereby the lymphocytes respond in the SCM test to cancer-associated antigens but not to mitogens after the contact.
  • a second property is the property of decreasing the spontaneous in vitro toxicity of potentially SCMresponding lymphocytes toward the human myeloid cell line K 562 by at least 90 percent as measured by 51 Cr release
  • Another aspect of the present invention is directed to methods of employing the general cancerassociated SCM factor in the SCM test.
  • this method comprises testing lymphocytes from a mammalian donor for the presence or absence of malignancy in the donor by: (1) contacting the lymphocytes with a general cancer-associated SCM-recognition factor; and (2) determining the decrease in the structuredness of the cytoplasmic matrix of the lymphocytes resulting from the step of contacting the suspension of the lymphocytes.
  • a preferred method for quantifying the decrease in structuredness comprises the steps of: (1) measuring the fluorescence polarization, P S , for an aliquot of the lymphocytes that has been contacted with the general cancer-associated SCM-recognition factor; (2) measuring the fluorescence polarization, P C , for a control aliquot of the lymphocytes that has not been contacted; and (3) determining the ratio of P S to P C .
  • a ratio of P S to P C lower than about 0.9 indicates a positive response to the SCM factor and the presence of a malignancy in the donor of the lymphocytes.
  • a more preferred method comprises comparing P S to the fluorescence polarization of another aliquot of the lymphocytes contacted with a mitogen, P M , to determine an SCM response ratio, RRSCM, where:
  • RR SCM P S /P M .
  • An RR SCM of less than about 0.9 indicates the presence of a malignancy in the donor of the lymphocytes.
  • the method of employing the SCM factor in the SCM test can comprise the steps of: (1) separating potentially SCM-responding lymphocytes from the blood sample; (2) contacting the separated lymphocytes with a general cancer-associated SCM-recognition factor thereby to stimulate the lymphocytes; (3) contacting the stimulated lymphocytes with a fluorogenic agent precursor, defined as a nonfluorogenic compound hydrolyzable intracellularly to a fluorogenic compound, to penetrate the lymphocytes for intracellular enzymatic hydrolysis to the fluorogenic compound, thereby generating stimulated fluor-containing lymphocytes; (4) exciting the stimulated fluorcontaining lymphocytes with polarized light thereby causing them to fluoresce; (5) measuring the vertically and horizontally polarized fluorescence emissions from the fluorescing lymphocytes for determining a polarization value for the fluorescing lymphocytes; and (6) comparing the determined polarization value for the stimulated fluor-containing lymphocytes with the polarization value for a control ali
  • the lymphocytes can be excited with vertically polarized light.
  • the polarization values when vertically polarized light is used are determined in a fluorescence spectrophotometer in accordance with the relationship:
  • I V and I H are the polarized fluorescence intensities in the vertical and horizontal planes, respectively; and G is a correction factor for the unequal transmission of the horizontal and vertical components of the polarized light through the optical system of the spectrophotometer.
  • the present invention provides a diagnostic technique for identifying subjects afflicted with cancer, it also comprehends methods for the treatment of such subjects. These methods are based on the observation that the general cancer-associated SCM-recognition factor has the property of decreasing the spontaneous in vitro toxicity of lymphocytes toward malignant cells. In light of that observation, the reduction of the in vivo activity of the SCM-recognition factor should increase the efficiency of immunological surveillance by lymphocytes against malignant cells.
  • this treatment is for a patient at least one of whose body fluids contains a general cancer-associated SCM-recognition factor.
  • the treatment comprises: (1) treating a body fluid containing the SCM-recognition factor to reduce the in vivo effect of the factor by selectively inactivating it; and (2) returning the body fluid to the patient, thereby to enhance the resistance of the patient to the cancer.
  • the body fluid is peripheral blood and the step of treating the body fluid comprises the dialysis of the peripheral blood to remove peptides with an apparent molecular weight of less than 1000 daltons, whereby the SCM-recognition factor is selectively inactivated by its removal from the blood.
  • the step of treating the body fluid can comprise neutralizing the SCM-recognition factor in the body fluid with an antibody specific for the factor, or with Fab fragments of such an antibody, the Fab fragments being capable of univalent binding of the SCM- recognition factor.
  • the present invention also encompasses a method of imaging cancer cells.
  • This method comprises: (1) labeling an antibody to the general cancer-associated SCM-recognition factor with an imaging substance; and (2) utilizing the labeled antibody to image cancer cells.
  • the present invention further encompasses a method of directing an anti-cancer substance to cancer cells, comprising: (1) tagging an antibody to the general cancer-associated SCM-recognition factor with the anti-cancer substance thereby to direct the anticancer substance to the cancer cells; and (2) administering the tagged antibody to a cancer patient so that the tagged antibody can bind to the cancer cells of the patient.
  • the present invention still further encompasses a method of determining the level of general cancer-associated SCM-recognition factor in a body fluid using an antibody to the general cancer-associated SCM-recognition factor in an immunoassay.
  • the immunoassay can be a radioimmunoassay, a fluorescence immunoassay, an enzyme-linked immunosorbent assay, or a precipitation immunoassay such as a nephelometric or a turbidimetric assay.
  • Fluorogenic Agent Precursor A nonfluorogenic compound capable of being taken up by lymphocytes and converted intracellularly by hydrolysis into a fluorogenic compound, of which the example used herein is fluorescein diacetate or FDA.
  • Standard SCM Test An SCM test using 1.0 ml of a lymphocyte suspension at 6 x 10 cells/ml and 0.1 ml of the mitogen or antigen, with FDA as the fluorogenic agent precursor and using an excitation wavelength of 470 nm and an emission wavelength of 510 nm for fluorescence polarization measurements.
  • SCM-Recognition Factor Material exhibiting SCM activity and of such a state of purity that the overwhelming majority of other molecules with specific biological activity, including all proteins and larger peptides, are not present in the material.
  • Tryptic Peptide A peptide cleaved from a larger peptide by the action of the proteolytic enzyme trypsin, which breaks peptide chains after lysine or arginine residues.
  • This invention relates to our discovery and purification to substantial homogeneity of a peptide that is a general cancer-associated SCM-recognition factor.
  • the factor passes through filters with a nominal molecular weight cutoff of 1000 daltons, but is retained by filters with a nominal molecular weight cutoff of 500 daltons.
  • the factor has an approximate amino acid composition of (Asx 2 , Glx 3 , Ser, His, Gly 5 , Thr, Arg, Ala 3 , Tyr, Met, Val 3 , Phe 3 , Ile, Leu 3 , Lys 2 ). Two active tryptic fragments have been isolated from different preparations of the factor. These fragments do not include the amino termini of the original factor molecules.
  • the first of these fragments has fifteen amino acids with the sequence Phe-Asn-Lys-Pro-Phe-Val-Phe-Leu-Met-Ile-Asp-Gln-Asn-Thr-Lys.
  • the second of these fragments has sixteen amino acids with the sequence Phe-Asn-Lys-Pro-Phe-Val-Phe-Leu-Met-Ile-Asp-Gln-Asn-Phe-Ser-Lys.
  • the factor Properties of the factor will be described, including the ability of the factor to modify the SCM response of lymphocytes from donors free of malignancy and to decrease the in vitro cytotoxicity of such lymphocytes toward malignant cells, as well as the crossreactivity of the factor isolated from donors with many different types of cancer.
  • a method for purification of this factor to substantial homogeneity will be described, as well as methods of using this factor in the SCM test.
  • Also to be described will be methods for treatment of cancer patients by reducing the in vivo activity of the factor, a method for imaging cancer cells by using antibody to the factor, and a method for directing anti-cancer substances to cancer cells by tagging antibody to the factor with the anticancer substances .
  • the amino acid composition of the general cancer-associated SCM-recognition factor purified from plasma of patients with breast cancer was determined, and the results are shown in Table 1. Lyophilized samples of the factor purified by RP-HPLC were hydrolyzed for 24 hr at 110°C with 2% thioglycolic acid in 6 N HCl in sealed glass ampules under nitrogen. After hydrolysis the samples were lyophilized. The residues were dissolved with 2% sodium dodecyl sulfate in 0.04 M sodium borate buffer. The amino acids were determined as o-phthaldialdehyde derivatives by their fluorescence after separation by HPLC as described in the article by B.N. Jones, S. Paabo and S.
  • the amino acid composition was determined after hydrolysis of dried peptides with 2% thioglycolic acid in 6 N HCl in sealed glass ampules under nitrogen. After hydrolysis samples were lyophilized and the residue dissolved with 2% SDS in 0.04 M sodium borate buffer. The amino acids were determined as o-phthaldialdehyde derivatives by their fluorescence as described in the manual, "Amino Acid Analysis by HPLC,” Altex Division, Beckman Instruments, Inc. Berkeley, CA 94710. The amino acids Cys and Pro were not determined.
  • the amino acid composition data of Table 1 show that the active factor is a peptide.
  • the data are most consistent with a peptide with the approximate amino acid composition (Asx 2 , GIX 3 , Ser, His, Gly 5 , Thr, Arg, Ala 3 , Tyr, Met 2 , Val 3 , Phe 3 , Ile, Leu 3 , Lys 2 ).
  • the amino acid sequences of segments isolated from two different preparations of the SCM factor were determined by performing automated Edman degradation procedures using an Applied Biosystems 477A protein sequencer coupled with an on-line 120A PTH-amino acid analyzer.
  • an Applied Biosystems 477A protein sequencer coupled with an on-line 120A PTH-amino acid analyzer.
  • Such fragments were obtained from separate purified preparations of the factor by tryptic digestion followed by purification of the tryptic fragments by reverse phase high pressure liquid chromatography, as detailed below under "Examples.”
  • One of these fragments was obtained from a preparation of the factor from plasma of patients with lung cancer, the other from plasma of patients with breast cancer.
  • the sequence calling computer program of the sequencer identified a sequence of sixteen amino acids as the sequence of the fragment of the SCM factor isolated from the plasma of lung cancer patients.
  • the sequence was: Phe-Asn-Lys-Pro-Phe-Val-Phe-Leu-Met-Ile-Asp-Gln-Asn-Phe-Ser-Lys.
  • the computer program identified a sequence of fifteen amino acids for the fragment of the factor isolated from the plasma of breast cancer patients. The sequence was: Phe-Asn-Lys-Pro-Phe-Val-Phe-Leu-Met-Ile-Asp-Gln-Asn-Thr-Lys.
  • the first thirteen amino acids in these two tryptic fragments are identical. Although their carboxyl termini are different, if the one missing amino acid, Phe, is inserted in the shorter fragment, the only difference then becomes the substitution of Thr for Ser, a very conservative substitution unlikely to affect the function of the peptide.
  • the purified SCM factor is fully active in the SCM test when used as a challenging agent for lymphocytes isolated from patients with several different types of malignancies. This activity can be demonstrated by assay at any point during the purification of the factor. Details of the results of such assays are given hereinbelow under "Examples.” The greatest activity is obtained with material taken through a final purification step, reverse phase high pressure liquid chromatography.
  • One-tenth milliliter of this fraction having an approximate protein content of 40 picomoles of peptide, causes decreases in intracellular fluorescence polarization of as much as 44.6% when used to challenge SCM-responding lymphocytes isolated from cancer patients, but causes no decrease in intracellular fluorescence polarization when used to challenge the same subpopulation of lymphocytes isolated from healthy donors.
  • This sequence is not the only sequence of thirteen amino acids believed to possess SCM activity. It is a well-established principle of protein and peptide chemistry that certain amino acid substitutions, entitled “conservative" amino acid substitutions, can frequently be made in a peptide without altering either the conformation or the function of that peptide. Such changes affecting amino acids in the core sequence include Ile and Leu for Val and vice versa. Asp for Glu and vice versa, and Asn for Gln and vice versa. Accordingly, it is believed that altered peptide molecules having the core structure in which any or all of the above-described conservative amino acid substitutions are made exhibit SCM activity and accordingly are considered part of the invention disclosed herein.
  • the lung and breast fragments are both isolated as part of larger general cancer-associated SCM-recognition molecules. Such fragments apparently can retain their SCM activity when incorporated into larger molecules. Therefore a larger peptide incorporating the thirteen-amino-acid core sequence, with or without the conservative amino acid substitutions discussed above, is also expected to exhibit SCM activity. The occurrence of these two types of modifications of the core sequence accordingly gives rise to an important additional aspect of the invention.
  • This is an SCM-active factor comprising only peptides of defined sequence, the peptides either having been shown directly to have SCM activity or believed to have such activity.
  • a factor comprises substantially only peptides having at least thirteen amino acid residues including a sequence of Phe-R 1 -Lys- Pro-Phe-R 2 -Phe-R 3 -Met-R 4 -R 5 -R 6 -R 7 therein, where R 1 is selected from the group consisting of Asn and Gln, R 2 , R 3 , and R 4 are each independently selected from the group consisting of Val, Leu, and Ile, R 5 is selected from the group consisting of Asp and Glu, and R 6 and R 7 are each independently selected from the group consisting of Asn and Gln.
  • this factor contains substantially only one such peptide.
  • the first class of these factors is derived from the sequence of the breast fragment, Phe-Asn-Lys-Pro-Phe-Val-Phe-Leu-Met-Ile-Asp-Gln-Asn-Thr-Lys.
  • This class most broadly comprises substantially only peptides having at least fifteen amino acid residues including a sequence of Phe-R 1 -Lys-Pro-Phe-R 2 -Phe-R 3 -Met-R 4 -R 5 -R 6 -R 7 -R 6 -Lys therein.
  • R 1 through R 7 are as defined above and reflect possible amino acid substitutions in the core sequence
  • R 8 is selected from the group consisting of Ser and Thr.
  • this factor contains substantially only one peptide.
  • a more clearly defined factor comprises substantially only peptides including a sequence of Phe-Asn-Lys-Pro-Phe-Val-Phe-Leu-Met-Ile-Asp-Gln-Asn-Thr-Lys. This is the original sequence of the breast fragment.
  • These peptides can include additional flanking sequences on either side of the specified sequence, and these flanking sequences can be of indeterminate length.
  • this factor also contains substantially only one peptide.
  • the most closely defined factor within this first class comprises a substantially pure peptide consisting essentially of the amino acid sequence Phe-Asn-Lys-Pro-Phe-Val-Phe-Leu-Met-Ile-Asp-Gln-Asn-Thr-Lys. This is the exact sequence of the breast fragment with no flanking sequences.
  • the second class of these factors is derived from the sequence of the lung fragment, Phe-Asn-Lys-Pro-Phe-Val-Phe-Leu-Met-Ile-Asp-Gln-Asn-Phe-Ser-Lys.
  • This class most broadly comprises substantially only peptides having at least sixteen amino acid residues including a sequence of Phe-R 1 -Lys-Pro-Phe-R 2 -Phe-R 3 -Met-R 4 -R 5 -R 6 -R 7 - Phe-R 6 -Lys therein.
  • R 1 through R 7 are as defined above and reflect possible amino acid substitutions in the core sequence
  • R 8 is selected from the group consisting of Ser and Thr.
  • this factor contains substantially only one peptide.
  • a more clearly defined factor comprises substantially only peptides including a sequence of Phe-Asn-Lys-Pro-Phe-Val-Phe-Leu- Met-Ile-Asp-Gln-Asn-Phe-Ser-Lys. This is the original sequence of the lung fragment.
  • These peptides can include additional flanking sequences on either side of the specified sequence, and these flanking sequences can be of indeterminate length.
  • this factor also contains substantially only one peptide.
  • the most closely defined factor within this second class comprises a substantially pure peptide consisting essentially of the amino acid sequence PheAsn-Lys-Pro-Phe-Val-Phe-Leu-Met-Ile-Asp-Gln-Asn-Phe-Ser-Lys. This is the exact sequence of the lung fragment with no flanking sequences.
  • the factor is designated as a general cancerassociated SCM factor because lymphocytes isolated from donors with all types of cancer respond to the factor in the SCM test.
  • the type of cancer afflicting the donor of the lymphocytes need not be the same as the type of cancer afflicting the donor of the body fluid from which the SCM factor was purified.
  • purification of the factor from blood plasma obtained from patients with nineteen different types of cancer always resulted in a factor eluting in the same position in reverse phase high-pressure liquid chromatography, strongly suggesting that the same molecule or a very similar molecule was isolated from all these patients.
  • amino acid sequencing of a tryptic fragment from the SCM factors isolated from blood plasma of patients with lung cancer and breast cancer shows that the first thirteen amino acids of the fragments are identical.
  • the only difference between the two sequences is the deletion of a phenylalanine residue present in position 14 of the lung fragment and the conservative substitution of threonine for serine in the next position. Details on the demonstration of the cross-reactivity of the SCM factor are given hereinbelow, under "Examples.”
  • the general cancer-associated SCM factor has the property of being able to modify the response of potentially SCM-responding lymphocytes obtained from donors free of malignancy when those lymphocytes are contacted with the factor.
  • the lymphocytes Before contact, the lymphocytes respond only to mitogens in the SCM test and do not respond to cancer-associated antigens.
  • the SCM response of the cells is modified to respond only to cancer-associated antigens and not to mitogens.
  • contact by such lymphocytes with the SCM factor alters their response in the SCM test from the normal response of lymphocytes from donors free of malignancy to the response seen with lymphocytes from donors afflicted with cancer. Details on the demonstration of this modification of the SCM response of lymphocytes from donors free of malignancy are given hereinbelow under "Examples.”
  • the SCM factor has the property of greatly decreasing the in vitro spontaneous natural cytotoxicity of potentially SCM-responding lymphocytes.
  • the general cancerassociated SCM recognition factor molecules are involved in the defense of cancer cells against the attack of killer lymphocytes and in this way help the survival and unrestrained growth of cancer cells.
  • the importance of the normal functioning of the immune system in controlling the growth of cancer cells is shown by the frequent occurrence of unusual forms of cancer in patients undergoing immune suppression. An important example of this is the occurrence of aggressive forms of Kaposi's sarcoma, ordinarily a rather slowly-spreading cancer, in patients afflicted with acquired immunodeficiency syndrome (AIDS).
  • AIDS acquired immunodeficiency syndrome
  • the general cancer-associated SCM factor modifies the SCM responses of lymphocytes from healthy donors and decreases the natural cytotoxicity of killer lymphocytes toward myeloid cells
  • treatment of a body fluid containing the SCM-recognition factor to reduce the in vivo effect of the factor by selectively inactivating it, followed by return of the body fluid to the patient can be used in management of cancer in order to increase the natural resistance to the disease.
  • the step of treating the body fluid can comprise the dialysis of the peripheral blood to remove peptides with an apparent molecular weight of less than 1000 daltons.
  • the step of treating the body fluid can comprise neutralizing the factor with an antibody specific for it, or with fragments of such an antibody, such as Fab fragments.
  • Fab fragments are capable of univalent binding so that each fragment molecule only binds one molecule of factor. This could be preferable to the use of intact antibody in some applications if the formation of large factor-antibody complexes is considered undesirable. This use of the general cancerassociated SCM cancer-recognition factor is described more fully hereinbelow.
  • the first step in purification of the SCM factor is obtaining an ultrafiltrate from a body fluid of a donor afflicted with cancer.
  • the body fluid can be peripheral blood, blood plasma, or urine; if the fluid is peripheral blood, the blood must be centrifuged to separate the red blood cells from the plasma.
  • the donor of the body fluid used for isolation of the SCM factor can be either autologous or allogeneic with respect to the lymphocytes used for the SCM test.
  • the ultrafiltration process separates a first fraction of the body fluid comprising molecules having an apparent molecular weight greater than 1000 daltons from a second fraction comprising molecules having an apparent molecular weight less than 1000 daltons.
  • the general cancer-associated SCM factor of the present invention is found in the second fraction of the ultrafiltrate.
  • the terms "apparent molecular weight” and "nominal molecular weight cutoff" are used herein because ultrafiltration is a somewhat imprecise method of separating molecules according to molecular weight in this molecular weight range, and the exact molecular weight excluded by a filter with a nominal molecular weight cutoff of 1000 daltons depends somewhat on the conformation of the molecule. Molecules larger than 1000 daltons in actual molecular weight can in fact pass through a filter with a nominal molecular weight cutoff of 1000 daltons if, for example, the molecules are relatively long and narrow.
  • the separation of the second fraction from the first fraction is performed by filtration of the body fluid through an ultrafilter with a nominal 1000-dalton molecular weight cutoff.
  • the purity of a preparation of such a factor, at the ultrafiltrate stage or later, can be described by its specific activity.
  • specific activity is defined as the reciprocal of the quantity of protein required to cause a particular degree of decrease, such as 20%, in the intracellular fluorescence polarization value when a particular fraction is used to challenge SCM-responding lymphocytes in the SCM test.
  • the goal of purification of the SCM factor is to increase the specific activity of the SCM factor over the specific activity found in the crude ultrafiltrate. The process of purification can therefore be followed by determining the specific activity of the purified fractions.
  • the protein concentration in the examples reported herein is only determined approximately in terms of ultraviolet absorbance, preferably at 220 nm, and the dose-response curve for the factor has not yet been determined, the characterization of various steps of the purification of the SCM factor described herein in terms of specific activity is only approximate. However, it is clear that the protein concentration decreases markedly as the factor moves through the various purification steps, while the activity is relatively unaffected, thereby resulting in an increase in specific activity of the SCM factor.
  • the ultrafiltrate can properly be described as consisting essentially of substantially pure general cancer-associated SCM-recognition factor, inasmuch as ultrafiltration through a membrane with a nominal molecular weight cutoff of 1000 will remove from a biological fluid the overwhelming majority of molecules with specific biological activity, including all proteins and larger peptides.
  • the next stage in the purification of the general cancer-associated SCM factor is a desalting step in which the second fraction obtained from ultrafiltration is loaded on a chromatographic column capable of separating the salts therefrom.
  • the material loaded onto the column is then eluted from the column with distilled water, and the portion eluting at an elution volume between about 0.3 and about 0.5 times the total chromatographic bed volume, containing the SCM factor, is collected.
  • the column used in this step is a gel filtration column with a fractionation range of from 0 to about 700 daltons, such as Sephadex (TM) G-10.
  • the next stage in the purification is another gel filtration step, again separating according to size.
  • the SCM-containing material obtained from the desalting step is loaded onto another gel filtration column, with a fractionation range of from about 1500 daltons to about 30,000 daltons, such as Sephadex (TM) G-50.
  • the material loaded onto the column is then eluted therefrom with a weak aqueous solution of an ammonium salt.
  • the ammonium salt is ammonium bicarbonate, more preferably 50 mM ammonium bicarbonate. That portion eluting at an elution volume between about 0.4 times and about 0.6 times the total chromatographic bed volume contains the SCM factor and is collected.
  • the next stage in the purification is an anion-exchange chromatography step, separating by charge.
  • the SCM factor-containing material from the previous gel filtration step is loaded onto an anionexchange column, preferably diethylaminoethyl cellulose (DEAE cellulose).
  • the material loaded onto the column is then eluted from the column with an increasing concentration of an ammonium salt.
  • ammonium salt is ammonium bicarbonate and the increasing concentration of the ammonium salt is from 10 mM to 1.0 M ammonium bicarbonate.
  • the fraction eluting from the column at about 0.28 M to about 0.31 M ammonium bicarbonate contains the SCM factor and is collected.
  • the final .stage of purification is reverse phase high pressure liquid chromatography (RP-HPLC), which separates by charge and/or hydrophobicity.
  • RP-HPLC reverse phase high pressure liquid chromatography
  • the SCM factor-containing material from the DEAE-cellulose column eluate is loaded onto a Aquapore RP-300 RP-HPLC column with dimensions of 220 mm x 2.1 mm. Elution is then performed with a combination of two solvents, initially 90 volume percent of 0.1 volume percent aqueous trifluoroacetic acid (TFA) and 10 volume percent of 0.09% volume percent of TFA in aqueous 70 percent acetonitrile, followed by a gradient with an increasing concentration of the acetonitrile-containing solution.
  • TFA trifluoroacetic acid
  • the SCM factor from all starting materials elutes as a homogeneous peak at a solvent composition of 26 volume percent of 0.1 volume percent aqueous trifluoroacetic acid and 74 volume percent of 0.09 volume percent aqueous trifluoroacetic acid in aqueous 70% acetonitrile.
  • RP-HPLC can be performed on a Beckman Ultrasphere ODS (TM) reverse phase high pressure liquid chromatography column. With this column, elution is then performed with a somewhat different solvent combination, initially 70 volume percent of 0.1 volume percent aqueous trifluoroacetic acid and 30 volume percent of 0.1 volume percent aqueous trifluoroacetic acid in aqueous 70% acetonitrile, followed by a gradient with an increasing concentration of the acetonitrilecontaining solvent.
  • TM Beckman Ultrasphere ODS
  • the SCM factor always elutes as an homogeneous peak at a solvent composition of 43.7 volume percent of 0.1% aqueous trifluoroacetic acid and 56.3 volume percent of 0.1% aqueous trifluoroacetic acid in aqueous 70% acetonitrile when this column and this solvent system is used.
  • the activity of the SCM factor is confirmed by its effect on viable potentially SCM-responding lymphocytes in accordance with the general test method described in the prior publication by L. Cercek and B. Cercek, "Application of the Phenomenon of Changes in the Structuredness of Cytoplasmic Matrix (SCM) in the Diagnosis of Malignant Disorders: a Review," Europ. J. Cancer 13., 903-915 (1977).
  • the general cancerassociated SCM-recognition factor of the present invention produces a significant decrease in the intracellular fluorescence polarization value of potentially SCM-responding lymphocytes from donors afflicted with cancer when used to challenge such lymphocytes in the SCM test performed as described in that article.
  • the degree of decrease of the intracellular fluorescence polarization value of such challenged lymphocytes is substantial-at least 20% even if ultrafiltrate is used to challenge such lymphocytes, and more than 50% if the most purified HPLC fraction is used.
  • lymphocytes The isolation of potentially SCM-responding lymphocytes is described in the European Journal of Cancer review article, supra, and also in a prior patent application by B. Cercek & L. Cercek, Serial No. 838,264, filed March 10, 1986, entitled "Automated Collection of Buoyant Density Specific Cells from Density Gradients," and incorporated herein by this reference.
  • the separation of these lymphocytes from the general lymphocyte population is important for the proper performance of the SCM test, because only a relatively small fraction of lymphocytes, approximately 20 to 25 percent, is capable of responding in the SCM test. Therefore, to perform the test on unfractionated lymphocytes results in a much smaller observed decrease in the intracellular polarization value even when the lymphocytes actually capable of responding in the SCM test fully responded.
  • a sample of peripheral blood is drawn from a donor and collected in a heparinized tube. After collection the peripheral blood is treated with iron powder or carbonyl-iron powder and the tubes containing the blood-iron powder mixture are placed on a magnet to effect separation of the phagocytic cells along with the iron powder from the blood sample. A portion of the blood sample is then transferred to a Ficoll (TM) -Triosil (TM) density gradient solution and centrifuged to effect separation of the potentially SCM-responding lymphocytes based on density differences.
  • TM Ficoll
  • TM Ficoll
  • TM Ficoll
  • TM Ficoll
  • TM Ficoll
  • TM Ficoll
  • TM Ficoll
  • TM Ficoll
  • TM Ficoll
  • TM Ficoll
  • TM Ficoll
  • TM Ficoll
  • TM Ficoll
  • TM Ficoll
  • TM Ficoll
  • TM Ficoll
  • TM Ficoll
  • Centrifugation is carried out at 550 x g for 20 min. at a temperature of 25° C.
  • the potentially SCM-responding lymphocytes are recovered using a Pasteur pipette to remove the cell layer separated above the density gradient material. Care must be taken to avoid, as much as possible, the removal of the density gradient material as this material includes various heavier plasma and cell components which interfere with the test results. Removal of the lighter plasma materials should also be avoided as much as possible to eliminate the introduction of any contaminants or SCM-nonresponding cells into the test samples.
  • the potentially SCM-responding lymphocytes are subjected to several washing steps, first in 0.9% preservative-free sodium chloride solution, then in complete Dulbecco's phosphate buffered saline (PBS) and held at 37° C for subsequent use in the SCM test procedure.
  • PBS Dulbecco's phosphate buffered saline
  • SCM-responding lymphocytes previously separated from the test subject's peripheral blood are incubated in sterile glass tubes at 37° C with a known concentration of either a mitogen such as phytohaemagglutinin or a cancer-associated antigen such as the general cancer-associated SCM-recognition factor which is the subject of the present invention.
  • a mitogen such as phytohaemagglutinin
  • a cancer-associated antigen such as the general cancer-associated SCM-recognition factor which is the subject of the present invention.
  • Other mitogens than phytohaemagglutinin (PHA), such as con ⁇ anavalin A and pokeweed mitogen, have been used, but PHA is preferred for the SCM test.
  • This incubation is initiated by adding 0.1 ml of the appropriately diluted mitogen or antigen to 1 ml of the cell suspension at 6 x 10 6 cells/ml. The incubation is then allowed to proceed for 30-60 min.
  • the incubated lymphocytes are then.admixed in suspension with a suitable nonfluorogenic compound hydrolyzable intracellularly to a fluorogenic compound, referred to hereinafter as a fluorogenic agent precursor, such as fluorescein diacetate (FDA).
  • a fluorogenic agent precursor such as fluorescein diacetate (FDA).
  • FDA fluorescein diacetate
  • the fluorescein diacetate is used at a final concentration of 2.5 mM in complete PBS at pH 7.4 and osmolality of 0.330 Osm/kg and is diluted from a concentrated stock solution prepared in acetone or glacial acetic acid. Aliquots of 0.2 ml of control or stimulated lymphocyte suspensions are slowly injected with a syringe into a beaker containing 3 ml of the FDA substrate solution.
  • the cells are exposed to the FDA for sufficient time (about 5 minutes) to allow for the penetration of the FDA substrate solution into the lymphocytes.
  • the nonfluorogenic fluorescein diacetate molecules are converted to fluorescein molecules by enzymatic hydrolysis.
  • the fluor-containing lymphocytes are isotropic in their response to polarized light since the polarization of the emitted fluorescence relative to that of the exciting light does not depend on the orientation of the plane-polarized light used to excite the lymphocytes.
  • the conventional fluorescence polarization measuring apparatus used herein for these measurements uses vertically polarized exciting light to excite the lymphocytes, so the measurement process is described in terms of vertically polarized exciting light.
  • the fluorescein molecules When exposed to excitation energy in the form of vertically polarized light, the fluorescein molecules emit fluorescence.
  • the relationship between the vertically polarized and horizontally polarized emissions is measured. This can be done by measuring the polarized fluorescence intensities in both the vertical and horizontal planes and determining a polarization value (P value) in accordance with the following relationship:
  • I V and I H are polarized fluorescence intensities in the vertical and horizontal planes, respectively, and G is a correction factor for the unequal transmission of the horizontal and vertical components of the polarized light through the optical system of the particular equipment used.
  • the P value of stimulated lymphocytes is compared with the P value of a control suspension of unstimulated lymphocytes from the same donor and the percent decrease in P value of the stimulated lymphocytes as compared to the P value of the control lymphocytes is an indication of the SCM-response to the cancer antigen.
  • the spectrophotometer utilized for SCM fluorescence measurements should be one of high sensitivity and stability and should be able to compensate for fluctuations in the intensity of the exciting light since the intensity of the polarized fluorescence emissions is recorded as a function of time and since the bulk concentration of fluorescein in the SCM measurements is only of the order of 10 -8 M to 10 -9 M. Also, broad band filter instruments are not suited for use for SCM measurements since SCM responses can be detected only within a narrow wavelength region.
  • the maximum spectral slit width of the excitation monochromator should be 20 nm and the maximum spectral slit width of the emission monochromator should be 10 nm when the excitation monochromator is set at 470 nm and the emission monochromator at 510 nm.
  • the spectrophotometer should also be fitted with a thermostatically controlled cuvette holder since the polarized fluorescent emissions are highly temperature dependent.
  • the spectrophotometer should also be provided with means for measuring both the horizontal and vertical polarized components of the fluorescent emissions.
  • the intensities of the emissions parallel to and perpendicular to the vertical exciting light beam are recorded alternately with an automatic polarizer changer for about 6 minutes or until the intensity of the emission perpendicular to the vertically exciting light beam reaches 80-90% of the full scale deflection of the recorder.
  • the cells are filtered away from the solution on a nitrocellulose filter of 0.22 ⁇ m pore size mounted in an appropriate filter head.
  • the fluorescence intensities parallel to and perpendicular to the exciting light are obtained.
  • the corrected fluorescein intensities for the cells are then obtained by subtracting the values obtained from the filtrate from the total fluorescence intensities extrapolated to the half time of filtration. This extrapolation is necessary because the background increases during the incubation because of the leakage of fluorescein from cells and spontaneous hydrolysis of FDA.
  • the general cancer-associated SCM factor whose purification is herein described can be used as an effective challenging agent in the SCM test for the detection of cancer.
  • cells from donors not afflicted with cancer exhibit no significant change in
  • SCM response when stimulated by cancer antigens used as challenging agents in the SCM test, while cells from donors afflicted with cancer exhibit a significant change in SCM response. This change is seen as a decrease of the P value when cells from donors afflicted with cancer are stimulated with cancer-associated antigens.
  • the SCM factor is nonspecific with respect to the type of cancer detected; the lymphocytes challenged with the SCM factor in the SCM test need not have come from a donor with the same type of cancer as the donor of the body fluid from which the SCM factor came.
  • the SCM factor When used as a challenging agent in the SCM test, the SCM factor produces a decrease of P value of at least ten percent, and can produce a decrease of P value of as much as 44.6%. If P S is the P value for an aliquot of the challenged lymphocytes, and P C is the P value for an aliquot of the lymphocytes not challenged with SCM factor, a ratio of P S to P C of less than about 0.9 is an indication of the presence of malignancy in the body of the donor of the challenged lymphocytes.
  • a preferred method of using the SCM factor as a challenging agent in the SCM test comprises comparing
  • RR SCM P S /P M .
  • An RR SCM of less than about 0.9 indicates the presence of a malignancy in the donor of the lymphocytes.
  • in vivo activity is meant the ability of the SCM-recognition factor to decrease the cytotoxicity of killer lymphocytes toward malignant cells.
  • a sample of a body fluid can be taken from the patient, passed through a filter with a nominal molecular weight cutoff of 1000 daltons, and the fraction passing through the filter collected and used to challenge lymphocytes from the same patient in the SCM test to confirm the presence of cancer-associated SCM-recognition factor in the fraction. It need not always be necessary to perform this test, particularly if other clinical indicators indicate the presence of cancer.
  • the body fluid can be treated by one of several methods to reduce the in vivo effect of the factor by selectively inactivating it, and the body fluid can then be returned to the patient, thereby enhancing the resistance of the patient to the malignancy.
  • the general cancerassociated SCM factor causes a response in the SCM test regardless of the type of cancer afflicting the patient, it follows that reducing the in vivo effect of the SCM factor can enhance the resistance of the patient not only to the particular type of cancer originally diagnosed, but also to any other type of malignancy which might subsequently develop. This can prove significant when treating patients with drugs that have an immunosuppressant effect, or patients with an already compromised immune system due to conditions such as AIDS.
  • one method of reducing the in vivo activity of the SCM factor is to physically remove it by dialysis of the peripheral blood to remove peptides with an apparent molecular weight of 1000 daltons or less, since the factor will pass through ultrafilters with a nominal molecular weight cutoff of 1000 daltons.
  • Another method of reducing the in vivo activity of the SCM factor in a body fluid is to neutralize it or inactivate it with antibody specific for the factor.
  • the SCM factor can be covalently coupled to a larger carrier molecule such as polylysine, and antibodies prepared to the conjugate, the SCM factor acting as a hapten. Once such antibodies are prepared, conventional methods can be used to isolate antibodyforming cells and produce monoclonal antibodies using cell fusion with hybridomas. Once antibody, whether polyclonal or monoclonal, is formed, it can be used to neutralize the SCM factor.
  • the antibody can be cleaved into univalent Fab fragments with the proteolytic enzyme papain.
  • Each fragment molecule produced by such cleavage can bind only one molecule of the factor, in contrast to intact antibody, which has sites for binding two separate molecules of factor.
  • Use of the fragments can be preferable to use of intact antibody in some applications if the formation of large factor-antibody complexes is considered undesirable.
  • An additional method of using antibodies produced to the SCM factor in the management of cancer is the tagging of the antibody with a substance with anti-cancer activity, in order to direct the anti-cancer substance to the site of the cancer and thereby raise the effective concentration of the anti-cancer substance at the site of the cancer. This procedure can be especially advantageous when the anti-cancer substance is one that produces side effects when given in larger doses.
  • the anti-SCM antibody can also be used to determine the levels of the SCM factor in peripheral blood or other bodily fluids by techniques such as radioimmunoassays, fluorescence immunoassays, or enzyme-linked immunosorbent assays, or by precipitation assay techniques such as nephelometry or turbidimetry, assuming that the SCM factor is not a univalent antigen. Assuming that the SCM factor is physically associated in some way with tumor cells, antibodies labeled with imaging substances such as fluorescent dyes or radioactive isotopes can also be used to image cancer cells so that cancer cells could be detected in biopsies. Also, fluorescent-labeled antibody can be used for automated detection of cancer cells by flow cytometry.
  • Blood samples from patients positively diagnosed as having active cancer such as cancer of the breast, lung, colon, ovary, cervix, uterus, larynx, or skin (basal cell carcinoma and malignant melanoma) were collected into heparinized vials such as Vacutainer (TM) tubes. Twenty-milliliter portions of the blood samples were centrifuged at about 1200 x g for approximately 40 min. The plasma above the sedimented blood cells was collected and filtered by pressure through a porous membrane filter such as an Amicon (TM) UM2 or YM2 filter, with a 1000-dalton molecular weight cutoff. These ultrafiltrates were lyophilized or stored at 4° until further purification.
  • TM Vacutainer
  • the data of Table 2 show that even when present in the crude ultrafiltrate, the SCM factor caused a decrease in the P value of the SCM-responding lymphocytes from donors afflicted with cancer at least equivalent to the decrease of the P values observed when lymphocytes are stimulated by crude extracts of cancerous tissues or cancerous tissues themselves.
  • the decrease in P value on stimulation by the ultrafiltrates was at least 10%, which is characteristic of such a positive SCM response.
  • the SCM factor did not pass through the Amicon (TM) UMO5 filter with a nominal 500-dalton molecular weight cutoff.
  • the lyophilized powder from the samples of Example 1 was dissolved in 2 ml of sterile preservativefree water for injections.
  • the SCM activity of the preparations was ascertained, and active samples from donors with the same type and site of cancer were pooled.
  • the pooled samples were desalted on an 0.9 x 18 cm column of Sephadex (TM) G-10, which has a fractionation range of from 0 to 700 daltons.
  • the sample volume per column chromatographic run did not exceed 25% of the column volume.
  • Elution was carried out with double distilled water at the linear elution speed of 8 to 9 cm/hr.
  • the desalting was carried out at room temperature (21°-23° C).
  • One-ml fractions eluting at between 0.3 and 0.5 times the total chromatographic bed volume were collected and the optical densities of the fractions determined.
  • the SCM activity was contained within the first elution peak. The presence of SCM activity in that peak was confirmed by an SCM test.
  • An aliquot of the first elution peak, prepared from an ultrafiltrate originally derived from plasma of a patient with breast cancer reduced the P value of lymphocytes from a patient with breast cancer to 86.3% of the control value in the SCM test, indicating the presence of SCM activity.
  • These fractions were collected and lyophilized.
  • the eluate was further purified by fractionation on a SephadeX (TM) G-50 gel filtration column, which has a fractionation range of from 1500 to 30,000 daltons.
  • the lyophilized desalted samples were dissolved in 50 mM NH 4 HCO 3 , loaded at no more than 5% of the column volume on a 0.9 x 18 cm Sephadex G-50 column at the linear elution speed of 3 cm/hr.
  • the elution was carried out at room temperature, and one-milliliter fractions eluting from the column at between 0.4 and 0.6 times the total chromatographic bed volume were collected. These fractions were tested for SCM activity. Results of these tests are given below in Example 4.
  • the SCM-active fractions were contained within the first elution peak as determined by optical densities of the one-milliliter fractions after testing of the fractions in the SCM test.
  • the active fractions from the same cancer types were pooled and lyophilized.
  • the lyophilized samples were dissolved in 10 mM NH 4 HCO 3 and loaded at no more than 4% of the column volume on an 0. 8 x 26 cm column of Whatman DE-52 microgranular DEAE-cellulose.
  • the column was washed with 10 ml of 10 mM aqueous NH 4 HCO 3 increasing by 0.108% per minute from 10 mM to 1 M NH 4 HCO 3 .
  • One-milliliter fractions were collected and the optical absorption at 220 nm was determined for each fraction. Based on the optical absorbance, active fractions eluting from the column at between 4.5 and 4.7 times the total chromatographic bed volume were pooled and lyophilized for testing and further purification. Results from SCM testing of the active fractions are given in Example 4.
  • Table 3 shows the results when aliquots of the Sephadex G-50 fractions from Example 3 originally from donors with various types of cancer were used to challenge lymphocytes from donors, also with various types of cancer, in the SCM test. It can be seen that potentially SCM-responding lymphocytes have the same characteristic response to the G-50 fractions as they did to the previously characterized cancer-associated antigens.
  • This desalted partially purified proteinaceous material exhibits a generally increased SCM response as compared to the crude ultrafiltrate for which the results were shown in Table 2. This increased SCM response is shown by decreased P values.
  • Diagnosis of Diagnosis of SCM Response Lymphocyte Donor SCM Factor Donor P Value as % of Control
  • Table 4 shows the results when SCM factor obtained from donors with various types of malignancies after purification through the DEAE-cellulose stage was used to challenge lymphocytes isolated either from donors with various types of malignancies or from donors free of malignancy in the SCM test.
  • the lymphocytes from cancer patients responded to the SCM factors purified from the DEAE-cellulose columns with a considerable decrease in P value, while lymphocytes from donors free of malignant disease showed no such decrease in P value.
  • Diagnosis of Diagnosis of SCM Response Lymphocyte Donor SCM Factor Donor P Value as % of Control
  • Example 4 The DE-52 general cancer-associated SCM-active fractions of Example 4 were then reconstituted and purified to homogeneity by reverse phase high pressure liquid chromatography (RP-HPLC) using a 2.1 mm x 22 cm HPLC column. The column was packed with Aquapore RP-300 (TM) (7 microns).
  • RP-HPLC reverse phase high pressure liquid chromatography
  • the mobile phases used in the RP-HPLC purification step were as follows:
  • Phase A 0.1 volume percent aqueous trifluoroacetic acid (TFA).
  • Phase B 0.09 volume percent aqueous TFA in aqueous 70% acetonitrile.
  • Lyophilized DE-52 SCM-active fractions were reconstituted with sterile water for injections (without preservatives) and 250 microliter aliquots were injected into the RP-HPLC column.
  • the mobile phase flow rate was 50 microliters per minute and its composition profile was 10 minutes of 90 volume percent of Phase A, 10 volume percent of Phase B, followed by 30 minutes of linear increase of Phase B at the rate of 3 volume percent per minute.
  • the optical density peaks detected by optical absorbance at 220 nm were hand-collected via a "nanobore" teflon tubing into 1.5 ml plastic conical Eppendorf centrifuge tubes and the solvent was evaporated in a vacuum centrifuge. In all cases, the general cancer-associated SCM-recognition factor eluted from the column at 74 volume percent of Phase B.
  • the SCM factor can be purified by performing HPLC using a 4.6 mmx 25 cm HPLC column packed with Ultrasphere ODS (TM) (5 microns) distributed by Beckman Instruments, Inc. with the DEAE-52 SCM-active fractions of Example 4.
  • TM Ultrasphere ODS
  • the mobile phases used with this column were as follows:
  • Phase A 0.1 volume percent aqueous trifluoroacetic acid (TFA).
  • Phase B 0.1 volume percent TFA in aqueous 70% acetonitrile.
  • the peptides were reconstituted with sterile water for injections without preservatives.
  • the SCM activity of SCM-responding lymphocytes after incubation with these samples is shown in Table 5. This fraction gives the greatest decrease in polarization value when used to challenge lymphocytes from donors afflicted with cancer.
  • Two of the three preparations of SCM factor grave a decrease in polarization value greater than 40%, a larger decrease than seen with any other fraction tested.
  • the purified factor as expected, was nonspecific with respect to the type of cancer afflicting the donor of the lymphocytes used. Also as expected, the purified factor gave no response when used to challenge lymphocytes from healthy donors.
  • Diagnosis of Diagnosis of SCM Response Lymphocyte Donor SCM Factor Donor P Value as % of Control
  • a sixteen-amino-acid long tryptic fragment was isolated from the purified SCM factor of Example 5. This fragment was shown to have the full SCM activity of the larger peptide, and was sequenced by the automated Edman degradation procedure. The cleavage of the purified SCM factor with trypsin and purification of the active fragment was carried out by the following procedure:
  • the SCM factor was digested with trypsin in the presence of HPLC eluants. Trypsin digestion was carried out in 0.1 M Tris-HCl buffer, pH 8.3, at 37° for 24 hours using 10 percent by weight of trypsin. The digest was diluted fourfold with 0.1 volume percent aqueous trifluoroacetic acid, and was injected into an Applied Biosystems 130A microflow HPLC-separation system. The tryptic fragments were separated using an Aquapore RP-300 column (200 mm x 2.1 mm). For the elution of the fragments, the mobile phase solvents were:
  • Phase A 0.1 volume percent aqueous trifluoroacetic acid (TFA).
  • Phase B 0.09 volume percent TFA in aqueous 70% acetonitrile.
  • the mobile phase flow rate was 50 ⁇ l per minute and the composition profile was 10 minutes of 96 volume percent Phase A, 4 volume percent Phase B, followed by a linear elution gradient comprising a 30 min linear increase in Phase B at a 3 volume percent per minute rate.
  • the SCM- active tryptic peptide fragment eluted at 69.6 volume percent of Phase B and 30.4 volume percent of Phase A in a total volume of about 30 microliters.
  • Table 6 summarizes the results obtained by using preparations of the general cancer-associated SCM recognition factor at various stages of purification from Examples 1, 3, and 6 as the challenging agent in the SCM test.
  • lymphocytes from donors afflicted with a number of different malignancies were used with the factor of the present invention in the SCM test, a significant response was seen in all cases.
  • This response is given in Table 6 as a percent of the control polarization value obtained by performing the SCM measurement on the same lymphocytes unincubated with the factor. The smaller the value the greater the response to the factor in the SCM test.
  • the ultrafiltrate give a decrease in polarization value of from 18.0% to 37.1%, and the most highly purified fraction, purified by RP-HPLC, gave a decrease in polarization value of as great as 44.6%.
  • the factor of the present invention is specific and only causes a decrease in polarization value when used to challenge lymphocytes from donors afflicted with cancer. Even the RP-HPLC purified fraction caused no decrease in polarization value when used to challenge lymphocytes from healthy donors.
  • the tryptic peptide whose purification was described above in Example 8 is fully active in the standard SCM test. Approximately 5 x 10 -2 femtograms of this fragment (i.e., 5 x 10 -17 grams, or approximately 16,000 molecules of the fragment) gave full activity in the test. When the fragment was isolated from patients with lung cancer, it proved active in the SCM test when tested against lymphocytes from a patient with small cell lung carcinoma, and crossreacted fully with lymphocytes from a patient with adenocarcinoma of the breast, as shown in Table 7. However, no response was seen when lymphocytes from healthy donors were used.
  • the following example demonstrates the ability of the SCM factor to cause a response in the SCM test when used to challenge lymphocytes derived from donors afflicted with dissimilar types of cancer.
  • two milliliters of cell-free blood plasma was obtained from each of a number of blood samples from cancer patients.
  • the blood samples had originally been collected into heparinized Vacutainer (TM) tubes.
  • the samples were ultrafiltered through an Amicon UM2 or YM2 filter with a nominal molecular weight cutoff of 1000 daltons for at least 12 hr and stored under sterile conditions at 4°C.
  • Potentially SCM-responding lymphocytes were isolated from heparinized blood samples from patients with cancer, healthy donors, and donors with non-malignant diseases.
  • SCM-responding lymphocytes were isolated from the blood samples of healthy donors and suspended in complete Dulbecco's phosphate buffered saline (PBS) at 5 x 10 5 cells/ml as described in the European Journal of Cancer article, supra, and also in the prior patent application by B. Cercek, Serial No.
  • PBS Dulbecco's phosphate buffered saline
  • the ability of the SCM factor to modify the SCM response of the lymphocytes from healthy donors was demonstrated by determining the SCM response ratio (RR SCM ) of the lymphocytes before and after the incubation with each of the fractions described above. Before being contacted with either a mitogen or with the SCM factor for determination of the RR SCM the incubated cells were thoroughly washed. The presence or absence of modification was determined by the ratio of the polarization value of the lymphocyte suspension after a short contact period with substrates containing the SCM factor over the polarization value of the lymphocyte suspension after a short contact period with phytohaemagglutinin (PHA).
  • PHA phytohaemagglutinin
  • Table 9 indicates the effect of the incubation with SCM-factor-containing fractions on the response of the lymphocytes to either SCM factor or phytohaemagglutinin, as reflected in the RR SCM.
  • Lymphocytes which were either not preincubated, or which were preincubated with ultrafiltrate from healthy donors filtered, through a filter with a nominal 1000-dalton molecular weight cutoff, or which were preincubated with ultrafiltrate from donors with cancer filtered through a filter with a nominal 500-dalton molecular weight cutoff, showed an RR SCM of 1.35 or higher, as expected.
  • lymphocytes which were preincubated with fractions containing SCM factor all showed decreases in the RR SCM to a value of 0.65-0.80 characteristic of lymphocytes originally isolated from patients with malignant disease.
  • lymphocytes obtained from healthy donors were incubated for 2 1/2 hr at 37°C with plasma containing SCM factor isolated as previously described from blood samples of donors afflicted with cancer. Aliquots of these lymphocytes were also retained as controls and not incubated.
  • potentially SCM-responding lymphocytes were obtained from donors having cancer, and treated in the same manner-some aliquots incubated with plasma containing SCM factor and others retained as controls and not incubated. After incubation the cytotoxicity of the lymphocytes was tested in accordance with the method described in M.R. Potter and M.
  • R S is the percent of 51 Cr release in the sample
  • Rc the percent of 51 Cr release in the control
  • R T is the percent of 51 Cr release i.n the presence of a detergent, Triton X-100.
  • Table 10 These results show that incubation of potentially SCM-responding lymphocytes from healthy donors for 2.5 hr with ultrafiltrates filtered through filter with a nominal 1000-dalton molecular weight cutoff decreased their cytotoxicity by over 90%. When the incubation was performed with potentially SCM-responding lymphocytes from cancer patients, the decrease in cytotoxicity was smaller, between 40 and 90%.
  • lymphocytes from cancer patients had lower levels of cytotoxicity before incubation, and the residual level of cytotoxicity remaining after incubation with ultrafiltrate was comparable to that remaining after incubation of lymphocytes from healthy donors.
  • the lower level of cytotoxicity present in cells from cancer patients was consistent with a decrease of such cytotoxicity caused by in vivo exposure to factors such as the general cancer-associated SCM recognition factor of the present invention.

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Abstract

On a isolé, purifié et caractérisé un facteur SCM général lié au cancer. Sont décrits des procédés d'application. Le facteur SCM est un peptide à faible masse moléculaire, pouvant passer à travers un filtre dont la masse moléculaire nominale-limite est de 1000 daltons, mais pas à travers un filtre dont la masse moléculaire nominale-limite est de 500 daltons. La composition approximative en amino-acides de ce facteur est (Asx2, Glx2, Ser, His, Gly5, Thr, Arg, Ala3, Tyr, Met, Val3, Phe3, Ile, Leu3, Lys2); il est capable de produire une baisse d'au moins 10% dans la valeur de polarisation de fluorescence de la fluorescine intracellulaire des lymphocytes susceptibles de réagir au SCM dans des prélèvements de sang de donneurs atteints du cancer. On a également purifié des peptides tryptiques de quinze ou seize amino-acides, non compris l'amino-terminus du facteur SCM: ils comportent une activité SCM. Le facteur SCM peut modifier la réponse au SCM de lymphocytes de donneurs qui ne sont pas atteints du cancer, qui devient une réponse caractéristique des lymphocytes des donneurs atteints du cancer. Ledit facteur peut en outre diminuer la cytotoxicité naturelle in vitro de lymphocytes tueurs envers les cellules tumorales. Est décrit un procédé pour purifier le facteur SCM du sang, qui produit, par chromatographie liquide à haute pression et phase inverse, un facteur purifié jusqu'à être substantiellement homogène. Le facteur SCM est utile, dans l'analyse de prélèvements sanguins, pour détecter la présence de tumeurs malignes chez le donneur par le test SCM. Des procédés pour limiter l'activité in vivo du facteur SCM, comme la dialyse ou la neutralisation d'anticorps, peuvent également être utiles pour enrayer le cancer.
EP19890904468 1988-03-11 1989-03-09 Facteur general de detection de scm lie au cancer; preparation et procede d'utilisation Withdrawn EP0407451A1 (fr)

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Publication number Priority date Publication date Assignee Title
US5580561A (en) * 1987-03-06 1996-12-03 Cercek; Boris Methods and pharmaceutical compositions for blocking suppression of immune defense mechanisms using an antibody, a factor, or an antisense peptide
US5270171A (en) * 1987-03-06 1993-12-14 Boris Cercek Cancer-associated SCM-recognition factor, preparation and method of use
US5516643A (en) * 1987-03-06 1996-05-14 Cercek; Boris Immunochemical assays for cancer-associated SCM-recognition factor
RU2065307C1 (ru) * 1994-12-13 1996-08-20 Александр Васильевич Тихонов Способ лечения первичного рака печени и набор для лечения первичного рака печени

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EP0260315B1 (fr) * 1986-03-10 1991-12-11 CERCEK, Boris Separation et procede d'utilisation de globules sanguins de densite specifique
US4835103A (en) * 1986-11-24 1989-05-30 Boris Cercek Differential binding of membrane potential sensitive materials to lymphocytes
AU624071B2 (en) * 1987-03-06 1992-06-04 Boris Cercek General cancer-associated scm-recognition factor, preparation and method of use

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See references of WO8908662A1 *

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