Yamamoto -- Gc-MAF vs Cancer **![](0logo.gif)** **[rexresearch.com](../index.htm)** --- **Nobuto YAMAMOTO** **GcMAF vs Cancer** --- **** **"Cancer Cured For Good" by Bill Sardi and Timothy Hubbell** **Video : -- GcMAF Explained --**[**http://video.google.com/videoplay?docid=2715490034719855134&pr=goog-sl**](http://video.google.com/videoplay?docid=2715490034719855134&pr=goog-sl) [**http://www.fritthelsevalg.org/htmlsite/aktuelt.asp?parent=1&fl**](http://www.fritthelsevalg.org/htmlsite/aktuelt.asp?parent=1&fl) "Serum vitamin D-binding protein - known as Gc protein - is the precursor of the principal macrophage activating factor," lead investigator Dr. Nobuto Yamamoto told Reuters Health. "Treatment of purified Gc protein with beta-galactosidase and sialidase generates Gc-MAF," he added, "the most potent macrophage activating factor ever discovered, which produces no side effect in humans." [**http://www.thedcasite.com/Yamamoto/Yamamoto\_biographical.html**](http://www.thedcasite.com/Yamamoto/Yamamoto_biographical.html) **Biographical Summary** Experimental and Molecular Therapeutics 11: Specific Immune Mechanisms and Cancer Vaccines: Clinical Studies Abstract #1255 Nobuto Yamamoto and Masumi Ueda Nobuto Yamamoto and Venkateswara R. Naraparaju / Cancer Res 1997 57: 2187-2192 **More Abstracts & Information : ** [**http://www.stopcancer.com**](http://www.stopcancer.com) 5/22/2008 **"Real Help for Cancer?" By Bill Sardi and Timothy Hubbell** --- **US5620846** **Preparation of potent macrophage activating factors derived from cloned vitamin D binding protein and its domain and their therapeutic usage for cancer, HIV-infection and osteopetrosis** **Abstract** -- Vitamin D-binding protein (Gc protein) and its small domain (approximately [1/5] of the Gc peptide also known as domain III) were cloned via a baculovirus vector. The cloned Gc protein and the cloned domain (Cd) peptide were treated with immobilized beta-galactosidase and sialidase to yield macrophage activating factors, GcMAFc and CdMAF, respectively. These cloned macrophage activating factors and GcMAF are to be used for therapy of cancer, HIV-infection and osteopetrosis, and may also be used as adjuvants for immunization and vaccination. Also published as: US 6410269 (B1) **Current U.S. Class**: 435/5 ; 435/18; 435/34; 435/974; 436/501 **Current International Class:** C07K 14/47 (20060101); C07K 14/435 (20060101); C12N 9/38 (20060101); G01N 33/574 (20060101); G01N 33/573 (20060101); G01N 33/569 (20060101); A61K 38/00 (20060101); A61K 39/00 (20060101); C12Q 001/70 () Field of Search: 435/4,5,18,34,974 436/501 **References Cited [Referenced By]** **Other References** Yagi et al, "Glycosidases of Ehrlich Ascites Tumor Cells and Ascitic Fluid-Purfication and Substrate Specificity of .alpha.-N-Acetylgalactosaminidase and .alpha.-Galactosidase: Comparison with Coffee Bean .alpha.-Galactosidase", Archives of Biochemistry and Biophysics, vol. 280, No. 1 (Jul. 1990), pp. 61-67. . Yamamoto et al, "Deglycosylation of Serum Vitamin D3-Biniding Protein Leads to Immunosuppression in Cancer Patients", Cancer Research, vol. 56, No. 12(1996 Jun. 15), pp. 2827-2831. . Yamamoto et al, "Structural Modification of Serum Vitamin D3-Binding Protein and Immunosuppression in AIDS Patients", AIDS Research in Human Retroviruses, vol. 11, No. 11(1995 Nov.), pp. 1373-1378. . Genomics, vol. 16, issued 1993, Witke et al., "Complet Structure of the Human Gc Gene: Differences and Similarities Between Members of the Albumin Gene Family", pp.751-754 see entire document. . Biochimica et Biophysica Acta, vol. 1216, issued 1993, Braun et al., "Sequence and Organization of the Human Vitamin D Binding Protein Gene", pp. 385-394, see entire document. . Proceedings of the National Academy of Science, U.S.A., vol. 82, issue Dec. 1985, Yang et al. "Human Group-Specifi Component (Gc) is a Member of the Albumin Family", pp. 7994-7998, see entire document. . Biochemica Et Biophysica Acta, vol. 871, issued 1986 Schoentgen et al., "Complete Amino Acids Sequence of Huma Vitamin D-Binding Protein (Group-Specific Component): Evidence of a Three-fold Internal Homology as a Serum Albumin an alpha-Fetoprotein", pp. 189-198, see entire document. . Ngwenya, B.Z., and Yamamoto, N. 1985. Activation of peritoneal macrophages by Iysophosphatidylcholine. Biochem. Biophys. Acta 839:9-15. . Ngwenya, B.Z., and Yamamoto, N. 1990. Contribution of Iysophosphatidyl-choline treated nonadherent cells to mechanism of macrophage stimulation. Proc. Soc. Exp. Biol. Med. 193:118-124. . Yagi, F., Eckhardt, A. E. and Goldstein I. J. 1990. Glycosidases of Ehrlich ascites tumor cells and ascitic fluid-purification and substrate specificity inidase and .alpha.-galactosidase: Comparison with coffee bean .alpha.-galactosidase. Arch. Biochem. Biophys. 280:61-67. . Yamamoto, N. and Homman, S. 1991. Vitamin D.sub.3 binding protein (group-specific component, Gc) is a precursor for the macrophage activating signal from sophosphatidylcholine-treated lymphocytes. Proc. Natl. Acad. Sci. USA. 88:8539-8543. . Yamamoto, N. and Kumashiro, R. 1993. Conversion of vitamin D.sub.3 binding protein (Group-specific component) to a macrophage activating factor by stepwise action of .beta.-galactosidase of B cells and sialidase of T cells. J. Imunol. 151:2794-2902. . Homma, S., Yamamoto, M. and Yamamoto, N. 1993. Vitamin D binding protein (group-specific component, Gc) is the sole serum protein required for macrophage activation after treatment of peritoneal cells with lysophosphatidylcholine. Immunol. Cell Biol. 71:249-257. Yamamoto, N., Kumashiro, R., Yamamoto, M., Willett, N.P. and Lindsay, D. D. 1993. Regulation of inflammation-primed activation of macrophages by two serum factors, vitamin D.sub.3 -binding protein and albumin. Inf. Imm. 61:5388-5391. . Yamamoto, N., Willett, N. P. and Lindsay, D. D. 1994. Participation of serum proteins in the inflammation-primed activation of macrophages. Inflammation. 18:311-322. . Naraparaju, V. R. and Yamamoto, N. 1994. Roles of .beta.-galactosidase of B lymphocytes and sialidase of T lymphocytes in infammation-primed activation of macrophages. Immunol. Lett. 43:143-148.. **Description** **FIELD OF THE INVENTION** This invention relates to methods to detect immunosuppression in cancer and AIDS patients, particular to the absence or reduced precursor activity for macrophage activating factor due to the presence of a serum glycosidase derived from these diseases. **BACKGROUND OF THE INVENTION** **A. Immunosuppression Resulting from Loss of MAF Precursor Activity** Inflammation results in activation of macrophages. Cellular membrane damage and the inflammatory process result in the release of lysophospholipids. Administration into mice of small doses (5-20 .mu.g/mouse) of lysophosphatidylcholine (lyso-Pc) and other lysophospholipids induced a greatly enhanced phagocytic and superoxide generating capacity of macrophages (Ngwenya and Yamamoto, Proc. Soc. Exp. Biol. Med. 193:118, 1990; Yamamoto et al., Inf. Imm. 61:5388, 1993; Yamamoto et al., Inflammation. 18:311, 1994). This macrophage activation requires participation of B cells and T lymphocytes and a serum vitamin D binding protein (DBP; human DBP is known as group specific components or Gc). Activation of mouse peritoneal macrophages by lyso-Pc requires modification of the Gc protein by stepwise association with .beta.-galactosidase of lyso-Pc-treated B cells and sialidase of T cells, to generate the macrophage activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety (FIG. 1a) (Yamamoto et al., Proc. Natl. Acad. Sci. USA. 88:8539, 1991; Yamamoto et al., J. Immunol. 151:2794, 1993). Thus, Gc protein is a precursor for MAF. Incubation of Gc protein with immobilized .beta.-galactosidase and sialidase generates a remarkably high titered MAF (GcMAF) (Yamamoto et al., Proc. Natl. Acad. Sci. USA. 88:8539, 1991; Yamamoto et al., J. Immunol. 151:2794, 1993; Naraparaju and Yamamoto, Immunol. Lett. 43:143, 1994; U.S. Pat. No. 5,177,002). Administration of a minute amount (10 pg/mouse; 100 ng/human) of GcMAF resulted in a greatly enhanced phagocytic capacity of macrophages. When peripheral blood monocytes/macrophages of 175 cancer patients bearing various types of cancer were treated in vitro with 100 pg GcMAF/ml, monocytes/macrophages (phagocytes) of all cancer patients were activated for phagocytic and superoxide generating capacity. This observation indicates that patient phagocytes are capable of being activated. However, the MAF precursor activity of plasma Gc protein was severely reduced in approximately one third of the cancer patient population. Loss of the MAF precursor activity prevents generation of MAF. Therefore, macrophage activation cannot develop in certain cancer patients. Since macrophage activation is the first step in immune development cascade, such cancer patients become immunosuppressed. This may explain at least in party why cancer patients die with overwhelming infections. About one third of the patients had moderately reduced MAF precursor activities while the remaining one third of the cancer patients had MAF precursor activities similar to those of healthy humans. Lost or reduced precursor activity of Gc protein was found to be due to deglycosylation of plasma Gc protein by .alpha.-N-acetylgalactosaminidase detected in a cancer patient's blood stream. Deglycosylated Gc protein cannot be converted to MAF (FIG. 1b). The source of the .alpha.-N-acetylgalactosaminidase appeared to be cancerous cells. Radiation therapy of cancerous lesions decreased plasma .alpha.-N-acetylgalactosaminidase activity with concomitant increase of precursor activity. This implies that radiation therapy decreases the number of cancerous cells capable of secreting .alpha.-N-acetylgalactosaminidase. Thus, plasma .alpha.-N-acetylgalactosaminidase activity has an inverse correlation with the MAF precursor activity of Gc protein. Both .alpha.-N-acetylgalactosaminidase activity and MAF precursor activity of Gc protein in a patient's blood stream can serve as diagnostic and prognostic indices. Similarly, when peripheral blood monocytes/macrophages of 65 HIV-infected/AIDS patients were treated in vitro with 100 pg GcMAF/ml, the monocytes/macrophages of all patients were activated for phagocytic and superoxide generating capacity. However, the MAF precursor activity of plasma Gc protein was severely reduced in about 1/10 of the HIV-infected patient population and approximately 25% of AIDS patients. These patients' plasma Gc protein is deglycosylated by .alpha.-N-acetylgalactosaminidase detected in HIV-infected patients. HIV-infected cells appeared to secrete .alpha.-N-acetylgalactosaminidase. Thus, .alpha.-N-acetylgalactosaminidase activity and MAF precursor activity of Gc protein in the patient's blood stream can serve as diagnostic and prognostic indices. In my prior two U.S. Pat. Nos. 5,177,001 and 5,177,002, the entire disclosures of which are incorporated by reference herein, as are my above cited journal articles, is disclosed various macrophage activating factors, processes for preparing them as well as methods of inducing macrophage activation in a person in need of such activation. **B. The Origin of .alpha.-N-acetylgalactosaminidase** Loss of the precursor activity was found to be due to deglycosylation of plasma Gc protein by .alpha.-N-acetylgalactosaminidase detected in the patient blood stream. The source of the enzyme appeared to be cancerous cells. Ehrlich ascites tumor cells contain a large amount of .beta.-N-acetylglucosaminidase and a very small amount of .alpha.-N-acetylgalactosaminidase (Yagi et al., Arch Biochem Biophys. 280:61, 1990). My data has indicated that both .beta.-N-acetylglucosaminidase and .alpha.-N-acetylgalactosaminidase were detected in tumor tissue homogenates as represented by enzyme activities (about 41.5 and 32.1 nmole/mg/min, respectively) of a lung tumor tissue. Similar results were also observed with eleven different tumor tissues including 4 lung, 3 breast, 3 colon and 1 cervix tumors, though the .alpha.-N-acetylgalactosaminidase activity varied from 5.9 to 50.8 nmoles/mg/min. Radiation therapy of cancerous lesions decreased plasma .alpha.-N-acetylgalactosaminidase activity with concomitant increase of precursor activity. This implies that radiation therapy decreases the number of cancerous cells capable of secreting .alpha.-N-acetylgalactosaminidase. Similarly HIV-infected patients carry .alpha.-N-acetylgalactosaminidase activity in their blood stream. HIV-envelope protein was found to contain .alpha.-N-acetylgalactosaminidase activity. HIV-infected cells can secrete this enzyme in to blood stream, resulting in deglycosylation of Gc protein. This would cause immunosuppression in HIV-infected/AIDS patients. Thus, both .alpha.-N-acetylgalactosaminidase activity and MAF precursor activity of Gc protein in patient blood stream can serve as excellent diagnostic and prognostic indices. **SUMMARY OF ASSAY PROCEDURES FOR PLASMA .alpha.-N-ACETYLGALACTOSAMINIDASE ACTIVITY AND MAF PRECURSOR ACTIVITY OF Gc PROTEIN:** 1. Precursor activity of vitamin D-binding protein (Gc protein) in patient plasma/serum for the macrophage activating factor. Assay procedure for precursor activity of serum (Gc protein) of cancer patient and HIV-infected/AIDS patients. **Step I. Lysophosphatidylcholine (Lyso-Pc)-treatment of mouse peritoneal cells (mixture of lymphocytes and macrophages):** Lyso-Pc (1 .mu.g/ml)+mouse peritoneal cells {30 min incubation at 37.degree. C. }.fwdarw.{washed with PBS} Step II. Lyso-Pc-treated peritoneal cells+gammaglobulin-depleted patient plasma/serum (0.1%) {3 hr cultivation at 37.degree. C.}.fwdarw.macrophage activation assay. {assay of superoxide generation} Precursor activity estimation: nanomoles of superoxide produced/min/10.sup.6 cells with patient plasma/serum compared with that of healthy human plasma/serum. 2. Detection of .alpha.-N-acetylgalactosaminidase in blood stream of cancer and HIV-infected/AIDS patients. Detection procedure for deglycosylating enzyme of serum Gc protein, .alpha.-N-acetylgalactosaminidase, in cancer patient and HIV-infected/AIDS patient blood stream. **Step I. Stepwise 30/70% ammonium sulfate precipitation of patient plasma/serum:** Patient plasma/sera (1 ml)+30% and 70% saturated ammonium sulfate 70% precipitate.fwdarw.dissolved in 50 mM citrate phosphate buffer (pH 6.0).fwdarw.dialyzed against the same buffer at 4.degree. C. for overnight. **Step II. Enzyme assay of .alpha.-N-acetylgalactosaminidase** Reaction mixture: 100 .mu.l of the dialyzed sample+1.0 ml of 50 mM citrate phosphate buffer (pH 6.0) containing 5 .mu.moles of p-nitrophenyl N-acetyl-.alpha.-D-galactosaminide as substrate. Incubation time: 60 min, terminated by adding 200 .mu.l of 0.5M Na.sub.2 CO.sub.3. Activity measurement: absorbance of amount of released p-nitrophenol at 420 nm and expressed as nmoles/min. **DESCRIPTION OF THE METHODS** **1. Precursor activity of serum Gc protein of cancer and HIV-infected/AIDS patients.** To determine precursor activity of Gc protein, mouse peritoneal cells (mixture of lymphocytes and macrophages) will be incubated with 1 .mu.g lysophosphatidylcholine (lyso-Pc)/ml in 0.1% egg albumin supplemented medium RPMI-1640 (EA medium) at 37.degree. C. for 30 min. The lyso-Pc-treated peritoneal cells will be washed with PBS and cultured for 3 h at 37.degree. C. in EA medium supplemented with gammaglobulin-depleted\* patient plasma/serum (0.1%) and assayed for superoxide generation of the macrophages. Loss or decrease of precursor activity of serum Gc protein results in lack or reduction of superoxide generation. Thus, the precursor activity is expressed by amounts of superoxide generated (nmoles of superoxide produced/min/10.sup.6 cells). **2. Assay procedure for .alpha.-N-acetylgalactosaminidase.** Plasma/serum (1 ml) of a healthy human and patients will be precipitated with 70% saturated ammonium sulfate. The ammonium sulfate precipitate will be dissolved in 50 mM citrate phosphate buffer (pH 6.0) and dialyzed against the same buffer at 4.degree. C. The volume of the dialysate will be made up to 1 ml and assayed for the enzyme. Ammonium sulfate precipitation is to separate the enzyme from inhibitors. The enzyme activity will be determined at 37.degree. C. in a reaction mixture of 1.0 ml containing 50 mM citrate phosphate buffer (pH 6.0) and 5 .mu.moles of p-nitrophenyl N-acetyl-.alpha.-D-galactosaminide as a substrate. The reaction will be initiated by addition of 100 .mu.l of the dialyzed samples and stopped after 60 min by adding 200 .mu.l of 0.5M Na.sub.2 CO.sub.3 solution. The reaction mixture will be centrifuged and amount of released p-nitrophenol will be determined by the absorbance of the supernatant at 420 nm and expressed as nmoles/min. **SUMMARY OF THE INVENTION** Cancerous cells and HIV-infected cells secrete .alpha.-N-acetylgalactosaminidase into the blood stream, resulting in deglycosylation of serum Gc protein. This inactivates the MAF precursor activity of Gc protein, leading to immunosuppression. Thus, both .alpha.-N-acetylgalactosaminidase activity and MAF precursor activity of Gc protein in patient blood stream can serve as diagnostic and prognostic indices. In one embodiment of the invention, the invention includes a process for determining macrophage activating factor precursor activity in plasma or serum of a person suspected of having cancer or HIV, comprising the step of quantifying in the plasma or serum an amount of vitamin D.sub.3 -binding protein. The determination of the macrophage activating factor precursor activity provides an indication of the patient's capability to activate its own monocytes/macrophages. In another embodiment of the invention, the invention includes a process for determining macrophage activating factor precursor activity in plasma or serum of a person suspected of having cancer or HIV comprising the step of quantifying in the plasma or serum an amount of .alpha.-N-acetylgalactosaminidase activity. Determining the .alpha.-N-acetylgalactosaminidase activity in the plasma or serum provides an indication of a quantity of malignant cells (or HIV-infected cells) in the plasma or serum. **DESCRIPTION OF THE DRAWINGS** Other objects and many attendant features of this invention will become readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein: **FIG. 1(a)** is a schematic illustration of the stepwise generation of macrophage activating factor. **FIG. 1(b)** is a schematic illustration of the stepwise deglycosylation of Gc protein. ![](fig1ab.jpg) **DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT** **II. Supporting Observations** Mechanisms of macrophage activation by lipid metabolites and a concept developed for therapy of immunodeficient diseases with vitamin D-binding protein derivatives are new and thus far have not been reported in the literature by others. Methods I have developed for diagnostic/prognostic indices are based on the following observations. **A) Cancer Patients** **1. Characterization of peripheral blood monocytes/macrophages, lymphocytes and Gc protein in cancer patients.** When peripheral blood monocytes/macrophages (phagocytes) of 175 cancer patients bearing various forms of cancer were treated with a small amount (100 pg/ml) of GcMAF, the phagocytes of all cancer patients were efficiently activated for production of more than 5.0 nmoles of superoxide produced/min/10.sup.6 cells, as can be seen at the last column of Table 1. When a mixture of nonadherent (B and T) lymphocytes and monocytes/macrophage (phagocytes) of individual patient was treated with 1 .mu.g lyso-Pc/ml for 30 min and cultured in a medium supplemented with 1 ng Gc protein/ml for 3 hr, the phagocytes of all cancer patients were efficiently activated, indicating that the B and T lymphocytes of all cancer patients are capable of generating macrophage activating factor (MAF) (more than 5.0 nmoles of superoxide produced/min/10.sup.6 cells, as can be seen in the 3rd column of Table 1). However, when the lyso-Pc-treated nonadherent and adherent cell mixtures of individual patients were cultured in medium supplemented with patient own plasma (0.1%) for 3 hr, the phagocytes of about 1/3 of the patients were not activated (less than 1.0 nmole of superoxide produced/min/10.sup.6 cells, as can be seen at the 2nd column of Table 1). These observations suggest that the patient B and T cells are capable of generating MAF while the MAF precursor activity of Gc protein in the plasma of 1/3 of this patient population was greatly reduced. With this assay procedure, another 1/3 patient population had moderately reduced precursor activity to support macrophage activation for generating 1.5-3.5 nmoles superoxide/min/10.sup.6 cells. The remaining cancer patients have precursor activity similar to those of healthy humans. Table 1 is exemplified by the data of the first 13 patients studied. Immunoblotting analysis of cancer patient and healthy human plasma revealed no quantitative change in Gc protein in cancer patients. Thus, lost or reduced precursor activity of Gc protein in certain cancer patients led us to suggest deglycosylation of Gc protein (FIG. 1b). Thus, inflammation-primed macrophage activation can not be developed in certain cancer patients. Since macrophage activation is the first step in the inflammation-primed immune development cascade, these cancer patients are immunosuppressed. This may explain at least in part why cancer patients die after resulted from overwhelming infection. TABLE 1 \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Activation of peripheral monocytes/macrophages by treatment with the enzymatically generated macrophage activating factor (GcMAF) or by treatment of mixture of nonadherent (B and T) lymphocytes and adherent cells (monocytes/macrophage s) with lysophosphatidylcholine (lyso-Pc) and followed by cultivation in media supplemented with purified Gc protein or plasma protein. Assay on lyso-Pc nmoles of superoxide produced/min/10.sup.6 cells +lymphocytes: none lymphocytes lymphocytes none Patient Cancer +phagocytes: phagocytes\* phagocytes phagocytes phagocytes\* No. type Protein: none 0.1% plasma 1 ng Gc 100 pg GcMAF \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ 1 Prostate ca.\*\* 0.10 0.56 5.69 6.13 2 Lung ca. 0.14 0.89 6.36 6.97 3 Prostate ca. 0.05 3.96 4.86 6.20 4 Lung ca. 0.25 0.80 5.04 5.19 5 kidney ca. 0.32 0.95 5.02 5.21 6 Lung ca. 0.21 1.99 5.32 5.82 7 Prostate ca. 0.29 7.44 6.73 7.47 8 Lung small cell ca. 0.88 1.74 6.24 6.89 9 Lung ca. 0.87 5.79 5.62 6.00 10 Pharynx/nasal sq. cell ca. 1.43 6.48 7.14 8.56 11 Cervix squamous cell ca. 0.35 6.46 6.32 8.03 12 Prostate ca. 0.61 1.98 6.43 7.04 13 Palate squamous cell ca. 0.65 5.28 6.51 10.08 C Healthy human 0.76 4.68 6.34 5.38 \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ \*phagocytes (monocytes/macrophages) were lysoPc-untreated. \*\*ca., carcinoma. Prostate ca., adenocarcinoma. The superoxide generating capacity of the phagocytes was expressed as nmoles of cytochromec reduced/min/10.sup.6 cells. TABLE 2 \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ N-acetylhexosaminidases detected in cancer patient peripheral blood and lung cancer tissue. N-acetylhexosaminidases.sup.a Source .alpha.-N-acetylgalactosaminidase .beta.-N-acetylglucosaminidase of Protein.sup.b total act. specific act. total act. specific act. Enzyme (mg) (.mu.moles/hr) (.mu.moles/mg/hr) (.mu.moles/hr) (.mu.moles/mg/hr) \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Normal plasma 68 0.94 .0138 90.37 1.329 Patient plasma 90 35.14 .3901 126.63 1.407 Lung tumor tissue\* 100 192.60 1.9260 249.04 2.490 \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ .sup.a N-acetylgalactosaminidase and N-acetylglucosaminidase activities are expressed as .mu.moles of nitrophenol production/hour from substrates pnitrophenyl Nacetyl-D-galactosaminide and pnitrophenyl Nacetyl-D-glucosaminide, respectively. .sup.b 70% ammonium sulfate precipitable protein of 1 ml samples of patient no. 1 and healthy human. \*1 g lung cancer tissue was homogenized in 3 ml (15 mM Tris buffer, pH 7) **2. Detection of N-acetylhexosaminidases in cancer patient plasma.** Electrophoretic analysis of patient plasma showed no quantitative change in Gc protein in these patient plasma. Thus, lost or reduced precursor activity of Gc protein in cancer patients suggests deglycosylation of Gc protein. Thus, deglycosylation of Gc protein in plasma may be due to the presence of N-acetylhexosaminidases in the blood stream (FIG. 1b). Patient and healthy human plasma were precipitated with 70% saturated ammonium sulfate. The precipitates were dialyzed and assayed for .alpha.-N-acetylgalactosaminidase and .beta.-N-acetylglucosaminidase using p-nitrophenyl N-acetyl-.alpha.-D-galactosaminide and p-nitrophenyl N-acetyl-.beta.-D-glucosaminide as substrates. Patients having lost or reduced precursor activity of plasma Gc protein carry a large amount of .beta.-N-acetylglucosaminidase and a significant amount of .alpha.-N-acetylgalactosaminidase while about the same amount of .beta.-N-acetylglucosaminidase and extremely low level (1/10) of .alpha.-N-acetylgalactosaminidase were found in healthy human plasma (Table 2). Since both healthy human and patient plasma contain the same .beta.-N-acetylglucosaminidase activity level, .beta.-N-acetylglucosaminidase may have nothing to do with deglycosylation of Gc protein. In fact, Gc protein is known to be O-glycosylated (43). When Gc protein as a macromolecular substrate and an equal amount (activity level) of .alpha.-N-acetylgalactosaminidase were used, the patient .alpha.-N-acetylgalactosaminidase deglycosylated Gc protein while healthy human enzyme was unable to deglycosylate Gc protein as shown in Table 3. This observation led us to conclude that the healthy human enzyme seems to be .alpha.-galactosidase simply because .alpha.-N-acetylgalactosaminidase and .alpha.-galactosidase share the same chromogenic substrate. Thus, .alpha.-N-acetylgalactosaminidase was identified in cancer patient blood stream exclusively. TABLE 3 \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Macromolecular substrate specificity of .alpha.-N-acetylgalactos- aminidase activity found in healthy human and cancer patient peripheral blood and lung cancer tissues. .alpha.-N-acetylgalactosaminidase.sup.a Enzyme Enzymatically treated Gc pro- Source Amount used tein used for precursor activity of total act. assay.sup.b Superoxide generated Enzyme (.mu.moles/hr) (nmoles/min/10.sup.6 cells) \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Normal plasma 0.24 4.09 Patient plasma 0.26 1.31 Lung tumor 0.24 1.33 tissue No enzyme None 4.02 \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ .sup.a N-acetylgalactosaminidase activity is expressed as .mu.moles of nitrophenol production/hour from substrate, pnitrophenyl Nacetyl-D-galactosaminide. .sup.b After 1 hr incubation of 1 ng Gc protein/ml with the indicated enzyme, the resultant product was added to lysoPc-treated mouse peritonea cells and cultured for 3 hr prior to superoxide generation assay of macrophages. **3. Detection of .alpha.-N-acetylgalactosaminidase in cancerous tissues.** Secretion of endo-.alpha.-N-acetylgalactosaminidase from tumor tissues is likely to be responsible for deglycosylation of Gc protein in the patient blood stream. Fresh post-operation tumor tissues were obtained and homogenized in 15 mM Tris Buffer at pH 7.0. The homogenates were treated with 70% ammonium sulfate for fractionation and the precipitate was dissolved in 50 mM citrate buffer at pH 4.5 and dialyzed in the same buffer at 4.degree. C. for overnight. Both .beta.-N-acetylglucosaminidase and .alpha.-N-acetylgalactosaminidase in tumor tissue homogenate were assayed. As shown in Table 2, large amounts of both .beta.-N-acetylglucosaminidase and .alpha.-N-acetylgalactosaminidase were detected in the tumor homogenates (data in Table 2 is exemplified by lung tumor). The latter enzyme was found to deglycosylate Gc protein (Table 3). It seems likely that secretory capacity of individual tumor tissue for N-acetylgalactosaminidase varies among cancer types. This would result in varying degrees of precursor activity of host plasma Gc protein. The extent of the decreased precursor activity may be reflection of invasiveness of tumor types. Thus, the precursor activity assay of individual patient should have diagnostic/prognostic utilities. **4. Effect of radiation therapy on the precursor activity of Gc protein.** As radiation therapy of cancer patients progressed, the majority of patients who initially had lost or decreased precursor activity of plasma Gc protein had a return toward or to normal (healthy human) values during radiation treatment (see the 2nd column of Table 4). This finding suggests that radiation therapy results in an increase in glycosylated Gc protein in peripheral blood. This also implies that radiation therapy decreases cancer cells capable of secreting .alpha.-N-acetylgalactosaminidase. This observation proved the precursor activity of patient Gc protein to be useful diagnostic/prognostic indices. TABLE 4 \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Time course study on the precursor activity of Gc protein for the macrophage activating factor in peripheral blood of cancer patients under radiation therapy and activation of peripheral monocytes/macrophages by treatment with GcMAF or by treat- ment of mixture of lymphocytes (B and T) and monocytes/ macrophages with lysophosphatidylcholine (lyso-Pc) and followed by cultivation in media supplemented with purified Gc protein or 0.1% patient plasma protein. nmoles of superoxide produced/min/10.sup.6 cells lyso-Pc lyso-Pc none Patient Day Treatment\*: none 0.1% 1 ng 100 pg No. assayed Protein: none plasma Gc/ml GcMAF \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ 1 Day 0 0.095 0.57 5.69 6.13 Day 7 0.197 0.88 5.20 5.65 Day 14 0.382 1.94 5.81 6.41 2 Day 0 0.142 0.89 6.36 6.97 Day 7 0.497 1.90 5.98 6.45 3 Day 0 0.247 3.97 4.86 6.20 Day 7 0.284 4.42 4.90 5.66 Day 14 0.541 6.27 6.55 8.04 5 Day 0 0.323 0.95 5.04 5.21 Day 7 0.309 0.98 5.47 5.79 Day 14 0.467 1.79 5.77 6.36 8 Day 0 0.875 1.74 6.24 6.89 Day 7 0.357 3.54 5.32 5.52 12 Day 0 0.612 1.98 6.43 7.01 Day 7 1.573 3.64 3.60 5.94 \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ \*Mixture of nonadherent (B and T) cells and adherent (monocytes/macrophages) was treated with 1 .mu.g lysoPc/ml for 30 min, washed with PBS and cultured for 3 hr in a medium supplemented with purified Gc protein or 0.1% patient plasma. **5. MAF precursor activity of Gc protein and .alpha.-N-acetylgalactosaminidase in oral cancer patient sera.** Among 175 cancer patients, we chose oral cancer patients for prolonged observation because of immediate perceptibility of tumor appearance and metastasis. As shown in Table 5, about 1/3 of total 18 patients exhibited greatly reduced precursor activity of serum Gc protein as expressed by less than 1.2 nmoles of superoxide produced/min/10.sup.6 cells. Another 1/3 of this patient population showed moderately reduced precursor activity, ranging for 1.5 to 3.5 nmoles of superoxide produced/min/10.sup.6 cells. The remaining patients had precursor activity level equivalent to that of healthy humans. Since loss of the precursor activity of serum Gc protein is resulted from deglycosylation of Gc protein by .alpha.-N-acetylgalactosaminidase, we assayed patient sera for .alpha.-N-acetylgalactosaminidase. As shown in Table 5, patients who had very low precursor activities carried very high enzyme activities in their blood stream. Patients who had high precursor activities carried very low enzyme activity. Thus, the enzyme activity levels of all patients showed an excellent inverse correlation with their precursor activity levels as can seen in Table 5. However, these immunological indices show no correlation with the degree of differentiation of tumors. About 50% of patients who had low precursor activities (less than 2.25) either were recurrent cases or developed metastasized lymph nodes during 6 month study period. Therefore, precursor activity of Gc protein and .alpha.-N-acetylgalactosaminidase activity in patient blood stream were proved to be excellent diagnostic/prognostic indices. TABLE 5 \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Histological analysis of squamous cell carcinoma, precursor activities of Gc protein and serum .alpha.-N-acetylgalactosaminidase (.alpha.-galNAc) of oral cancer patients. Patient Precursor activity\* .alpha.-galNAc No. Site Type nmoles of superoxide.sup..dagger. nmoles/mg/min \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ 1 Tongue, Verrucous carcinoma 2.61 1.80 2 Tongue, Well differentiated scc.dagger-dbl. 1.94 6.51 3 Oral floor, Well differentiated scc. 5.19 1.11 4 Upper gingiva, Moderately differentiated scc. 4.90 1.31 5 Oral floor, Well differentiated scc. 5.90 1.09 6 Tongue, Well differentiated scc. 4.66 1.20 7 Lower gingiva, Well differentiated scc. 1.07 3.51 8 Maxillary sinus, Adenoid cystic carcinoma 4.23 1.22 9 Lower gingiva, Well differentiated scc. 4.25 0.94 10 Maxillary sinus, Poorly differentiated scc. 2.24 2.42 11 Tongue, Well differentiated scc. 3.45 1.96 12 Upper gingiva, Well differentiated scc. 1.11 7.40 13 Maxillary sinus, Poorly differentiated scc. 2.31 3.02 14 Buccal mucosa, Well differentiated scc. 0.06 7.03 15 Tongue, Well differentiated scc. 1.19 7.42 16 Lower gingiva, Well differentiated scc. 2.14 3.12 17 Maxillary sinus, Well differentiated scc. 0.05 7.88 18 Lower gingiva, Well differentiated scc. 2.76 2.34 Healthy human 5.10 0.05 \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ \*Mixture of healthy human lymphocytes (B and T cells) and phagocytes (monocytes/macrophages) cells was treated with 1 .mu.g lysoPc/ml for 30 min, washed with PBS and cultured for 3 hr in a medium supplemented with 0.1% patient plasma. .sup..dagger. unit is nmoles of cytochromec reduced/min/10.sup.6 cells. .dagger-dbl.scc, squamous cell carcinoma. **B) HIV-infected/AIDS Patients** **1. Characterization of peripheral blood monocytes, macrophages and Gc protein in HIV-infected/AIDS patients.** When peripheral blood monocytes/macrophages (phagocytes) of 65 HIV-infected patients were treated with a small amount (100 pg/ml) of GcMAF, the phagocytes of all patients were activated for generating more than 4.0 nmoles of superoxide produced/min/10.sup.6 phagocytes as with healthy humans. When a mixture of lymphocytes and phagocytes of a healthy human was treated with 1 .mu.g lyso-Pc/ml for 30 min and cultured in a medium supplemented with 0.1% patient plasma for 3 h, the phagocytes were not activated with patient plasma of about 1/10 of the total patient population and produced less than 0.7 nmoles of superoxide/min/10.sup.6 phagocytes. These patients having severely decreased precursor activity were found to be approximately 1/4 of the AIDS patients. The plasma Gc protein of the majority (65%) of HIV-infected patients was capable of being converted to MAF as expressed by more than 4.0 nmoles of superoxide produced/min/10.sup.6 phagocytes while the MAF precursor activity of Gc protein in the plasma of approximately 25% of this patient population was moderately reduced (ranging 1.6-3.6 nmoles of superoxide produced/min/10.sup.6 phagocytes) as shown in Table 6. This observation suggests that the phagocytes of all HIV-infected patients are capable of being activated while the precursor activity of Gc protein for MAF in the plasma of certain AIDS patients was severely reduced. This may explain at least in part why AIDS patients die from overwhelming bacterial infection. **2. Detection of N-acetylgalactosaminidase in HIV-infected patient plasma.** Electrophoretic analysis of patient plasma showed no quantitative change in Gc protein in these patient plasma. Thus, lost or reduced precursor activity of Gc protein in HIV-infected/AIDS patients suggests deglycosylation of Gc protein. Deglycosylation of Gc protein in plasma was found to be due to the presence of endo-.alpha.-N-acetylgalactosaminidase in the patient blood stream. Patients having lost or reduced precursor activity of plasma Gc protein carried significantly large amounts of .alpha.-N-acetylgalactosaminidase activity in their blood stream while an extremely low level of .alpha.-N-acetylgalactosaminidase activity was detected in healthy human plasma. As shown in Table 7, the enzyme activity in patient plasma showed excellent inverse correlation with the precursor activity of the patient plasma Gc protein, confirming our hypothesis that .alpha.-N-acetylgalactosaminidase deglycosylates plasma Gc protein. However, the enzyme activity and CD4.sup.+ value of the patients showed no obvious correlation. When Gc protein as a macromolecular substrate was treated with an equal activity (4 nmoles/min) of the enzyme from patient and healthy human plasma, the patient .alpha.-N-acetylgalactosaminidase efficiently deglycosylated Gc protein thus inactivated the precursor activity while the healthy human enzyme was unable to deglycosylate Gc protein. The inability of the healthy human enzyme to catabolize Gc protein may imply that this activity in healthy human is .alpha.-galactosidase, because .alpha.-N-acetylgalactosaminidase and .alpha.-galactosidase are evolutionary related, carry 46.9% amino acid sequence homology and share common chromogenic substrate for their catabolic capacities. Thus, a significant amount of .alpha.-N-acetylgalactosaminidase was detected exclusively in HIV-infected/AIDS patient blood stream. TABLE 6 \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Activation of monocytes/macrophages (phagocytes) and precursor activity of plasma Gc protein of individual HIV-infected/AIDS patients. nmoles of cytochrome-c reduced/min/10.sup.6 cells Assayed phago- phagocytes lymphocytes/ Patient Stage on: cytes\* 100 pg phagocytes\*\* No. CD4.sup.+.sctn. Protein: none GcMAf 0.1% plasma \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ 1 115 0.29 5.68 6.01 2 445 0.25 4.84 4.74 3 516 0.67 5.12 5.39 4 188 0.41 4.11 0.54 5 102 0.36 4.02 3.42 6 136 0.67 4.04 0.69 7 577 0.29 7.52 4.43 8 160 0.42 4.29 5.14 9 222 0.87 4.21 5.22 10 156 0.61 4.98 5.03 11 441 0.35 4.48 4.69 12 298 0.10 7.22 4.52 13 849 0.14 6.50 3.14 14 56 0.56 5.04 4.32 15 22 0.84 4.32 2.91 16 418 0.71 4.33 4.08 17 721 0.61 4.05 4.41 18 989 0.44 4.26 4.04 19 585 0.38 4.01 3.62 20 64 0.45 4.73 4.33 21 845 0.08 4.85 2.91 22 326 0.29 4.82 1.64 23 305 0.52 4.61 4.63 Control.dagger. 0.54 5.01 5.10 \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ \*Phagocytes indicates monocytes/macrophages of patients. \*\*Precursor activity of plasma Gc protein as measured by superoxide generating capacity of the phagocytes after 3 h incubation of a mixture o lysoPc-treated lymphocyte and phagocytes of healthy human with 0.1% plasm of individual patients. .sup..sctn. CD4.sup.+ cell count per cubic mm. .dagger. Average of 5 healthy humans. TABLE 7 \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ .alpha.-N-acetylgalactosaminidase activity detected in HIV-infected patient plasma and its correlation with the precursor activity of plasma Gc protein and the CD4.sup.+ value of the patients. Patient .alpha.-N- plasma acetylgalactosaminidase Disease stage indices\* (Patient Specific activity CD4.sup.+ no.) (nmoles/mg/min) Precursor act. value \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Healthy 0.056 5.12 ND.sup..sctn. human 4 13.12 0.54 188 5 2.51 3.42 102 6 12.80 0.69 136 7 1.13 4.43 577 13 2.63 3.14 849 15 3.15 2.91 22 19 2.28 3.62 585 21 3.03 2.91 845 22 3.54 1.64 326 \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ \*These values were derived from Table 6. .sup..sctn. Not determined. References 1. Ngwenya, B. Z., and Yamamoto, N. 1985. Activation of peritoneal macrophages by lysophosphatidylcholine. Biochem. Biophys. Acta 839:9-15. 2. Ngwenya, B. Z. and Yamamoto, N. 1990. Contribution of lysophosphatidyl-choline treated nonadherent cells to mechanism of macrophage stimulation. Proc. Soc. Exp. Biol. Med. 193:118-124. 3. Yagi, F., Eckhardt, A. E. and Goldstein I. J. 1990. Glycosidases of Ehrlich ascites tumor cells and ascitic fluid-purification and substrate specificity inidase and .alpha.-galactosidase: comparison with coffee bean .alpha.-galactosidase. Arch. Biochem. Biophys. 280:61-67. 4. Yamamoto, N. and Homma, S. 1991. Vitamin D.sub.3 binding protein (group-specific component, Gc) is a precursor for the macrophage activating signal from sophosphatidylcholine-treated lymphocytes. Proc. Natl. Acad. Sci. USA. 88:8539-8543. 5. Yamamoto, N. and Kumashiro, R. 1993. Conversion of vitamin D.sub.3 binding protein (Group-specific component) to a macrophage activating factor by stepwise action of .beta.-galactosidase of B cells and sialidase of T cells. J. Immunol. 151:2794-2902. 6. Homma, S., Yamamoto, M. and Yamamoto, N. 1993. Vitamin D binding protein (group-specific component, Gc) is the sole serum protein required for macrophage activation after treatment of peritoneal cells with lysophosphatidylcholine. Immunol. Cell Biol. 71:249-257. 7. Yamamoto, N., Kumashiro, R., Yamamoto, M., Willett, N. P. and Lindsay, D. D. 1993. Regulation of inflammation-primed activation of macrophages by two serum factors, vitamin D.sub.3 -binding protein and albumin. Inf. Imm. 61:5388-5391. 8. Yamamoto, N., Willett, N. P. and Lindsay, D. D. 1994. Participation of serum proteins in the inflammation-primed activation of macrophages. Inflammation. 18:311-322. 9. Naraparaju, V. R. and Yamamoto, N. 1994. Roles of .beta.-galactosidase of B lymphocytes and sialidase of T lymphocytes in inflammation-primed activation of macrophages. Immunol. Lett. 43:143-148. Without further elaboration the foregoing will so fully illustrate my invention that others may, by applying current or future knowledge, adapt the same for use under various conditions of service. --- **http://www3.interscience.wiley.com/journal/116330149/abstract** ***International Journal of Cancer*, Volume 122, Issue 2, January 15, 2008, Pages: 461-467** **Immunotherapy of metastatic breast cancer patients with vitamin D-binding protein-derived macrophage activating factor (GcMAF)** **Nobuto Yamamoto, Hirofumi Suyama, Nobuyuki Yamamoto, Naofumi Ushijim** **Abstract --** Serum vitamin D3-binding protein (Gc protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of breast cancer patients was lost or reduced because Gc protein was deglycosylated by serum -N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Patient serum Nagalase activity is proportional to tumor burden. The deglycosylated Gc protein cannot be converted to MAF, resulting in no macrophage activation and immunosuppression. Stepwise incubation of purified Gc protein with immobilized -galactosidase and sialidase generated probably the most potent macrophage activating factor (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages treated in vitro with GcMAF (100 pg/ml) are highly tumoricidal to mammary adenocarcinomas. Efficacy of GcMAF for treatment of metastatic breast cancer was investigated with 16 nonanemic patients who received weekly administration of GcMAF (100 ng). As GcMAF therapy progresses, the MAF precursor activity of patient Gc protein increased with a concomitant decrease in serum Nagalase. Because of proportionality of serum Nagalase activity to tumor burden, the time course progress of GcMAF therapy was assessed by serum Nagalase activity as a prognostic index. These patients had the initial Nagalase activities ranging from 2.32 to 6.28 nmole/min/mg protein. After about 16-22 administrations (approximately 3.5-5 months) of GcMAF, these patients had insignificantly low serum enzyme levels equivalent to healthy control enzyme levels, ranging from 0.38 to 0.63 nmole/min/mg protein, indicating eradication of the tumors. This therapeutic procedure resulted in no recurrence for more than 4 years. --- ***Proc Amer Assoc Cancer Res*, Volume 45, 2004.** **Cancer cell-killing by macrophages treated with Gc protein-derived macrophage activating factor (GcMAF)** **Nobuto Yamamoto and Masumi Ueda** Abstract: Macrophages, when highly activated via inflammation (e.g. intratumor BCG administration), can eradicate cancerous cells. Inflammation-primed macrophage activation process is the principal macrophage activation cascade that requires serum Gc protein (vitamin D-binding protein) and participation of B and T lymphocytes. Stepwise hydrolysis of Gc protein with ss-galactosidase of inflammation-primed B cells and sialidase of T cells yields a potent macrophage activating factor (MAF), a Gc protein with N-acetylgalactosamine as the remaining sugar. Thus, Gc protein is the precursor for MAF. Stepwise treatment of purified Gc protein with immobilized ss-galactosidase and sialidase generates the most potent macrophage activating factor (GcMAF) ever discovered which produces no side effect in humans and animals. Macrophages activated by GcMAF (100 pg every 4 days to Ehrlich ascites tumor bearing mice) eradicate the tumor cells. After more than 25 weekly administrations of 100 ng GcMAF to cancer patients, the majority of prostate and breast cancer patients, excluding extremely advanced, exhibited healthy control levels of the tumor markers (e.g., -N-acetylgalactosaminidase), indicating eradication of tumors confirmed by CAT-scan and MRI. We demonstrated rapid in vitro cancer cell-killing using mouse and human macrophages activated by GcMAF. Since activation of macrophages leads to enhanced phagocytosis and antigen presentation to lymphocytes for development of humoral and cellular immunity, GcMAF therapy of Ehrlich ascites tumor-bearing mice and prostate and breast cancer patients appeared to develop antibodies against their respective cancerous cells. When in vitro cancer cell-killing study with mouse and human macrophages activated by GcMAF was performed using Ehrlich ascites tumor, and breast and prostate cell lines in the presence of serum (or IgG) fraction of GcMAF-treated Ehrlich tumor bearing mice, prostate or breast cancer patients, greatly accelerated cell-killings were observed, indicating that sera of GcMAF treated tumor-bearing mice and cancer patients contain IgG antibodies against these cancer cells because inflammation (or GcMAF)-primed activation of macrophages is known to develop Fc-receptor mediated cell-killing and phagocytosis of targets preferentially --- **** ***Journal of the National Cancer Institute*, Vol. 94, No. 17, 1311-1319, September 4, 2002** **Effects of Vitamin D3-Binding Protein-Derived Macrophage Activating Factor (GcMAF) on Angiogenesis.** **Shigeru Kanda, Yasushi Mochizuki, Yasuyoshi Miyata, Hiroshi Kanetake, Nobuto Yamamoto.** Conclusions: In addition to its ability to activate tumoricidal macrophages, GcMAF has direct antiangiogenic effects on endothelial cells independent of tissue origin. The antiangiogenic effects of GcMAF may be mediated through the CD36 receptor. --- **** **Intratumor induced inflammation generates maximally activated macrophages that can eradicate cancerous cells.** **Nobuto Yamamoto\*. Socrates Institute For Therapeutic Immunology, Philadelphia, PA.** Photodynamic action on mammalian tissues immediately results in severe inflammation, leading to macrophage activation. Intratumor PDT-induced inflammation generates maximally activated macrophages that can eradicate local as well as metastasized cancerous cells. Inflammation in mammalian tissues activates phospholipase A2 to releases lysophospholipids that efficiently activate macrophages. Because cancerous tissues contain alkylphospholipids, PDT-induced inflammation of cancerous tissue produces alkyl-lysophospholipids and alkylglycerols that activate macrophages with approximately 400 times more efficiency than lysophospholipids. These results imply that highly activated macrophages can kill cancerous cells. Inflammation-primed macrophage activation process is the principal macrophage activation cascade that requires serum vitamin D3-binding protein (known as Gc protein) and participation of B and T lymphocytes. Stepwise hydrolysis of Gc protein with the inducible membranous beta-galactosidase of inflammation-primed B cells and the membranous Neu-1 sialidase of T cells yields a potent macrophage activating factor (MAF), the protein with N-acetylgalactosamine as the remaining sugar. Thus, Gc protein is the precursor for the principal MAF. Stepwise treatment of highly purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage activating factor (termed GcMAF) ever discovered which produces no side effect in humans. Administration of 100 ng GcMAF per human and 100 pg GcMAF per mouse results in the maximal activation of macrophages, which develop enormous variation of receptors. When human macrophages were treated in vitro with GcMAF (100 pg/ml) for 3 hrs, the macrophages were highly activated. The activated macrophages can recognize and kill a variety of cancerous cells indiscriminately. When prostate and breast, cancer patients were treated with less than 25 weekly administrations of 100 ng GcMAF, the majority of cancer patients, excluding anemic patients, exhibited healthy control levels of the serum prognostic index, alpha-N-acetylgalactosaminidase, indicating eradication of the tumors. GcMAF therapy also develops antibodies against the tumors. --- **** **Cancer Research 57, 295-299, January 15, 1997.** **Prognostic Utility of Serum a-N-Acetylgalactosaminidase and Immunosuppression Resulted from Deglycosylation of Serum Gc Protein in Oral Cancer Patients.** **Nobuto Yamamoto, Venkateswara R. Naraparaju and Masahiro Urade.** **Abstract:** Vitamin D3-binding protein (Gc protein), a serum glycoprotein, is the precursor for the macrophage activating factor. Cancer patient sera contain -N-acetylgalactosaminidase that deglycosylates Gc protein. Deglycosylated Gc protein cannot be converted to macrophage activating factor, leading to immunosuppression. Of 46 oral cancer patients with squamous cell carcinoma, approximately 22% had greatly reduced precursor activities. The precursor activity of approximately 61% of these patients was moderately reduced. The remaining patients (17%) had precursor activities equivalent to those of healthy humans. Patients with low precursor activity of serum Gc protein had high serum -N-acetylgalactosaminidase activity. In contrast, patients with high precursor activity had low serum -N-acetylgalactosaminidase activity. Thus, levels of serum -N-acetylgalactosaminidase of individual patients have an inverse correlation with precursor activities of their serum Gc protein. Surgical removal of tumors resulted in a subtle decrease in serum -N-acetylgalactosaminidase activity with concomitant increase in the precursor activity of serum Gc protein. Serum enzyme analysis of nude mice transplanted with a human oral squamous carcinoma cell line revealed that serum -N-acetylgalactosaminidase activity is directly proportional to tumor burden. Thus, -N-acetylgalactosaminidase activity in patient blood-stream can serve as a diagnostic/prognostic index. --- **** ***Proceedings of the Society for Experimental Biology and Medicine*, Volume 220, Issue 1 Page 20-26 ( January 1999 )** **Antitumor Effect of Vitamin D-Binding Protein-Derived Macrophage Activating Factor on Ehrlich Ascites Tumor-Bearing Mice.** **Yoshihiko Koga, Venkateswara R. Naraparaju & Nobuto Yamamoto.** **Abstract**: Cancerous cells secrete ?-N-acetylgalactosaminidase (NaGalase) into the blood stream, resulting in deglycosylation of serum vitamin D3-binding protein (known as Gc protein), which is a precursor for macrophage activating factor (MAF). Incubation of Gc protein with immobilized ?-galactosidase and sialidase generates the most potent macrophage activating factor (designated GcMAF). Administration of GcMAF to cancer-bearing hosts can bypass the inactivated MAF precursor and act directly on macrophages for efficient activation. Therapeutic effects of GcMAF on Ehrlich ascites tumor-bearing mice were assessed by survival time and serum NaGalase activity, because serum NaGalase activity was proportional to tumor burden. A single administration of GcMAF (100 pg/mouse) to eight mice on the same day after transplantation of the tumor (5 x 105 cells) showed a mean survival time of 21 +/- 3 days for seven mice, with one mouse surviving more than 60 days, whereas tumor-bearing controls had a mean survival time of 13 +/- 2 days. Six of the eight mice that received two GcMAF administrations, at Day 0 and Day 4 after transplantation, survived up to 31 +/- 4 days whereas, the remaining two mice survived for more than 60 days. Further, six of the eight mice that received three GcMAF administrations with 4-day intervals showed an extended survival of at least 60 days, and serum NaGalase levels were as low as those of control mice throughout the survival period. The cure with subthreshold GcMAF-treatments (administered once or twice) of tumor-bearing mice appeared to be a consequence of sustained macrophage activation by inflammation resulting from the macrophage-mediated tumoricidal process. Therefore, a protracted macrophage activation induced by a few administrations of minute amounts of GcMAF eradicated the murine ascites tumor. --- [**http://pt.wkhealth.com/pt/re/imcb/abstract.00004228-199806000-00006.htm;jsessionid=LcWVyp7QF8ls1WyyM59wqYbL3gXWQ52mFRwGLQ3yhx1LTGGyqZgw!-1331995130!181195629!8091!-1**](http://pt.wkhealth.com/pt/re/imcb/abstract.00004228-199806000-00006.htm;jsessionid=LcWVyp7QF8ls1WyyM59wqYbL3gXWQ52mFRwGLQ3yhx1LTGGyqZgw%21-1331995130%21181195629%218091%21-1) ***Immunology & Cell Biology* 76(3):237-244, June 1998.** **Structurally well-defined macrophage activating factor derived from vitamin D3-binding protein has a potent adjuvant activity for immunization.** **YAMAMOTO, NOBUTO ; NARAPARAJU, VENKATESWARA R** Summary: Freund's adjuvant produced severe inflammation that augments development of antibodies. Thus, mixed administration of antigens with adjuvant was not required as long as inflammation was induced in the hosts. Since macrophage activation for phagocytosis and antigen processing is the first step of antibody development, inflammation-primed macrophage activation plays a major role in immune development. Therefore, macrophage activating factor should act as an adjuvant for immunization. The inflammation-primed macrophage activation process is the major macrophage activating cascade that requires participation of serum vitamin D3-binding protein (DBP; human DBP is known as Gc protein) and glycosidases of B and T lymphocytes. Stepwise incubation of Gc protein with immobilized [beta]-galactosidase and sialidase efficiently generated the most potent macrophage activating factor (designated GcMAF) we have ever encountered. Administration of GcMAF (20 or 100 pg/mouse) resulted in stimulation of the progenitor cells for extensive mitogenesis and activation of macrophages. Administration of GcMAF (100 pg/mouse) along with immunization of mice with sheep red blood cells (SRBC) produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days. Thus, GcMAF has a potent adjuvant activity for immunization. Although malignant tumours are poorly immunogenic, 4 days after GcMAF-primed immunization of mice with heat-killed Ehrlich ascites tumour cells, the ascites tumour was no longer transplantable in these mice. --- ****< Year 2008, Volume 1, Issue 2 **Immunotherapy of prostate cancer with Gc protein-derived macrophage activating factor, GcMAF** **Nobuto Yamamoto, Hirofumi Suyama and Hiroyuki Yamamoto** **Abstract --** Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage activating factor (termed GcMAF) ever discovered, which produces no side effect in humans. Macrophages activated by GcMAF develop a considerable variation of receptors that recognize the abnormality in malignant cell surface and are highly tumoricidal. Sixteen nonanemic prostate cancer patients received weekly administration of 100 nanogram (ng) GcMAF. As the MAF precursor activity increased their serum Nagalase activity decreased. Since serum Nagalase activity is proportional to tumor burden, the entire time course analysis for GcMAF therapy was monitored by measuring the serum Nagalase activity. After 14-25 weekly administrations of GcMAF (100 ng/week), all sixteen patients had very low serum Nagalase levels equivalent to those of healthy control values, indicating that these patients are tumor free. No recurrence occurred for seven years. --- **US6410269** **Diagnostic and Prognostic Indices for Cancer and Aids** Also published as: **US5620846** 1997-04-15 Classification: - international: C07K14/47; C12N9/38; G01N33/569; G01N33/573; G01N33/574; A61K38/00; A61K39/00; C07K14/435; C12N9/38; G01N33/569; G01N33/573; G01N33/574; A61K38/00; A61K39/00; (IPC1-7): C12Q1/70 :- European: C07K14/47; C12N9/38; G01N33/569K2; G01N33/573; G01N33/574V Also published as: US6410269 (B1) Abstract -- Cancerous cells and HIV-infected cells secrete alpha -N-acetylgalactosaminidase into the blood stream, resulting in deglycosylation of serum Gc protein. This inactivates the MAF precursor activity of Gc protein, leading to immunosuppression. Thus, both alpha -N-acetylgalactosaminidase activity and MAF precursor activity of Gc protein in patient blood stream can serve as diagnostic and prognostic indices. In one embodiment is disclosed a process for determining macrophage activating factor precursor activity in plasma or serum of a person suspected of having cancer or HIV, comprising the step of quantifying in the plasma or serum an amount of vitamin D3-binding protein. The determination of the macrophage activating factor precursor activity provides an indication of the patient's capability to activate its own monocytes/macrophages.; In another embodiment is disclosed a process for determining macrophage activating factor precursor activity in plasma or serum of a person suspected of having cancer or HIV comprising the step of quantifying in the plasma or serum an amount of alpha -N-acetylgalactosaminidase activity. Determining the alpha -N-acetylgalactosaminidase activity in the plasma or serum provides an indication of a quantity of malignant cells (or HIV) in the plasma or serum ---