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Role of Vitamin D Binding Proteinderived Macrophage Activating Factor (GcMAF) in the immunotherapy of cancer Lynda Thyer, Rod Smith, Emma Wards, David Noakes Macro Innovations Ltd.,Cambridge, UK Immuno Biotech Ltd, Guernsey, Channel Isles In modern times Immunotherapy was mentioned in 1971 in Science and now there are more than 56.000 papers on the subject. There has been much recent interest in the role of the vitamin D axis in the immunotherapy of cancer. The vitamin D axis includes vitamin D, vitamin D receptor (VDR), and vitamin D-binding protein (VDBP; also known as Gc-globulin) that is the precursor of the Gc globulin-derived Macrophage Activating Factor (GcMAF). GcMAF proved effective as an anticancer agent in a variety of experimental and spontaneous tumours. The first paper on GcMAF published in a peer-reviewed scientific journal indexed in the National Library of Medicine is dated 1994. About 20 years later the research on GcMAF is a solid scientific reality with tens of papers published on its effects in vitro and in vivo and with hundreds of presentations at international congresses describing its effects in a variety of experimental systems as well as in several pathologic conditions. In addition to stimulating macrophages that infiltrate tumour tissues, GcMAF directly inhibits human tumour cell proliferation in vitro and reverts the malignant phenotype of human breast cancer cells. Furthermore, GcMAF inhibits cancer cell-induced angiogenesis, thus depriving the tumour cell mass of the blood, oxygen and nutrient supply that it needs to grow and metastasize. We demonstrated that the effects of GcMAF are mediated by the vitamin D receptor (VDR), and we demonstrated that GcMAF stimulates the intracellular signalling pathway belonging to the cAMP cascade. This pathway could be responsible for the apoptosis of tumour cells induced by GcMAF. We recently demonstrated that these effects are due to the interaction GcMAF/vitamin D receptor (VDR). 12 Val 62 Glu 61 Glu 60 Ala 63 Ala 64 Asp 65 Pro

4 Gly 3 Arg 2 Glu 1 Leu NH2 22 Glu 23 Asp 24 Phe 25 Thr 26 Ser 27 Leu 28 Ser 29 Leu 30 Val 31 Leu 32 Tyr 33 Ser 34 Arg 35 Lys 51 Val 50 Glu 49 Lys 48 Val 47 Leu 46 Gln 45 Ser 44 Val

43 Gln 42 Gln 41 Phe 40 Thr 39 Gly 38 Ser 37 Pro 74 Ala 75 Leu 76 Ser 77 Ala 78 Lys 79 Ser 80 Cys 81 Glu 82 Ser 83 Asn 84 Ser 85 Pro 86 Phe 87 Pro 99 Glu 98 Lys 97 Thr 96 Cys 95 Cys 94 Glu

93 Ala 91 Gly 90 Pro 89 His 101 Leu 102 Glu 103 Arg 104 Lys 105 Leu 106 Cys 107 Met 108 Ala 116 Glu 115 Gln 114 Pro 113 Gln 112 His 111 Lys 100 Leu 109 Ala 11 Lys 10 Asp 9 Lys 8 Glu 7 Tyr 6 Asp 5

Arg 13 Cys 14 Lys 15 Glu 16 Phe 17 Ser 18 His 19 Leu 20 Gly 21 Lys 57 Ala 56 Glu 55 Thr 54 Leu 53 Leu 52 Val 70 Thr 71 Arg 72 Thr 73 Ser 59 Cys 66 Asp 58 Cys 67 Cys 68 Tyr 69 Asp 100 Gly Domain I 127

Glu 126 Asp 124 Thr 123 Pro 122 Glu 121 Val 120 Tyr 119 Thr 118 Pro 117 Phe 129 Cys 130 Glu 131 Ala 132 Phe 133 Arg 134 Lys 135 Asp 136 Pro 137 Lys 138 Glu 139 Tyr 140 Ala 141 Asn 142 Gln 143 Phe 144 Met 145 Trp

146 Glu 147 Tyr 148 Ser 149 Thr 150 Asn 151 Tyr 173 Cys 172 Ser 171 Gly 170 Val 169 Met 168 Ser 167 Leu 166 Tyr 165 Ser 164 Lys 163 Thr 162 Tyr 161 Ser 160 Val 159 Leu 158 Leu 157 Ser 156 Leu 155 Pro 154 Ala 153 Gln

152 Glu 183 Phe 184 Leu 185 Lys 186 Glu 187 Arg 188 Leu 189 Gln 190 Leu 191 Lys 216 Arg 217 Leu 218 Ser 219 Asn 220 Leu 221 Ile 222 Lys 223 Leu 224 Ala 225 Gln 226 Lys 234 Asp 233 Glu 232 Leu 231 Asp 230 Ala 229

Thr 274 Ser 275 Thr 276 Lys 278 Ser 176 Ser 175 Thr 174 Cys 178 Ser 179 Pro 180 Thr 181 Val 182 Cys 203 Val 202 Arg 201 Asn 200 Ser 199 Leu 198 Thr 197 Thr 196 Leu 195 Leu 194 Ser 193 Leu 204 Cys 205 Ser 206 Gln 207

Tyr 208 Ala 209 Ala 210 Tyr 211 Gly 212 Glu 213 Lys 214 Lys 248 Lys 247 Ser 246 Leu 245 Ile 244 Asn 243 Thr 242 Ile 241 Asp 240 Glu 239 Ala 238 Leu 237 Pro 236 Leu 235 Val 255 Gln 256 Asp 257 Cys 258 Met 259 Ala

260 Lys 261 Gln 262 Leu 263 Pro 264 Glu 265 His 266 Thr 267 Val 268 Lys 269 Leu 270 Cys 271 Asp 272 Asn 273 Leu 288 Thr 287 Lys 286 Glu 285 284 Glu Cys 283 Cys 282 Asp 281 Glu 280 Phe 279 Lys 290 Met 291 Asp 292 Val 293 Phe

294 Val 295 Cys 296 Thr 297 Tyr 298 Phe 299 Met 308 Pro 307 Leu 306 Glu 305 Pro 304 Leu 303 Gln 302 Ala 301 Ala Pro 330 Val 333 Glu 334 Leu 335 Ser 336 Arg 337 Arg 338 Thr 343 Val 342 Glu 341 Pro 340 Leu 339 His

410 Leu 411 Lys 412 Ala 413 Lys 316 Lys 315 Asn 314 Thr 318 Val 319 Cys 320 Asp 362 Val 361 Asp 360 Cys 359 Cys 365 Ser 366 Thr 367 Thr 368 Cys 389 Glu 391 Cys 390 Leu 440 Asn 441 Ser 442 Pro 227 Val 249 Cys 317 Asp

Domain III 215 Ser 250 Cys 289 Ala 364 Asp 192 His 251 Glu Domain II 363 Glu 88 Val 128 Ile 177 Ala 252 253 Ser Ala 254 Ser 125 Asn 92 Thr 36 Phe 313 Pro 321 Pro 312 Leu 311 Arg 310 Val 309 Asp 322 Gly 323 Asn 324 Thr 325 Lys 326 Val

327 Met 328 Asp 329 Lys 358 Glu 357 Gly 356 Lys 355 Ser 354 Lys 353 Leu 352 Thr 351 Pro 369 Phe 370 Asp 371 Ala 372 Lys 373 Gly 374 Pro 375 Leu 376 Leu 377 Lys 388 Gln 387 Gly 386 Lys 385 Asp 384 Ile 383 Phe 382 Ser

381 Ser 380 Leu 379 Glu 392 Ala 393 Asp 394 Tyr 395 Ser 396 Glu 397 Asn 398 Thr 399 Phe 400 Thr 401 Glu 350 Glu 331 Thr 332 Phe 349 Leu 348 Val 403 Lys 404 Lys 347 Lys 346 Ser 345 Leu 344 Phe 406 Leu 407 Ala 408

Glu 228 Pro 277 Asp 300 378 Lys 402 Tyr 439 Ile 438 Ser 437 Cys 436 Cys 435 Asn 434 Ser 433 Ala 432 Phe 431 Asp 430 Ser 429 Arg 428 Lys 427 Asn 426 Val 425 Leu 424 Lys 443 Pro 444 Leu 445 Tyr 446 Cys 447 Asp 448

Ser 449 Glu 450 Ile 451 Asp 452 Ala 453 Glu 454 Leu 455 Lys 456 Asn 457 Ile 458 Leu 405 Lys 423 Ala 422 Leu 421 Glu 420 Thr 409 Arg 419 Pro 418 Thr 417 Ala 416 Glu 414 Leu 415 Pro COOH Gc protein (known as Vitamin D3-binding protein) 420 thr A VDR GcMAF Vit D Vit D

cytoplasm GcMAF Oleic acid Plane of the membrane B Vit D Plane of the membrane VDR cytoplasm GcMAF Oleic Oleic acid acid GcMAF by interacting with the VDR modifies the expression of a number of genes in all the cell types expressing the VDR and elicits a series of responses that go well beyond its capability of activating macrophages. BAG gene has been implicated in age related neurodegenerative diseases as Alzheimer's Here I shall present results demonstrating that: 1. Macrophages activated by GcMAF search and destroy human breast cancer cells. 2. GcMAF, dissolved in a solvent designed to fit its molecular features, inhibits human breast cancer cell tumorigenic potential. 3. GcMAF reduces nagalase levels in patients with cancer and improves clinical conditions. Experimentals 1 Purified, activity-tested GcMAF was obtained from Immuno Biotech Ltd., Guernsey, Channel Islands. All other reagents were from Sigma Aldrich, Milano, Italy. In some experiments, GcMAF was dissolved in a hydrophobic/hydrophilic solvent designed to fit its molecular features Human breast cancer cells (MCF-7) were obtained from the Istituto Zooprofilattico Sperimentale della Lombardia e dellEmiliaRomagna, Brescia, Italy. In experiments of co-cultures, macrophages (Raw 264.7, HPA Culture Collection) were activated by culturing them in the same medium of MCF-7 cells and in the presence of 100 ng/mL GcMAF for 72 h prior to addition to the MCF-7 cell culture. The macrophages were added at a ratio of 1:1 to the MCF-7 cell culture. The cells were then allowed to settle for 1 h before time-lapse photography. Photography was taken over a 7-day period using an Olympus CK2 microscope and a GXCAM-3 with NCH Debut capture software. Experimentals 2 A retrospective chart review for analysis of nagalase testing was accomplished on the initial cohort of patients tested by the treating clinicians All records were reviewed by physicians for confirmation of test results, confirmed diagnoses, the time intervals between testing, the dosing of subsequent GcMAF used and the observed clinical responses. The oncologic diagnosis was confirmed by other treating physicians. Administration of GcMAF to individual patients was performed exclusively by their treating physicians (Robert Eslinger, MD, Reno Integrative Medical Center; Reno, NV, USA, and Steven Hofman, MD, CMC-Capelle a/d Ijssel, The Netherlands) according to the rules and regulations of each respective Country. The original clinical records are conserved by the physicians in their respective locations as indicated. Nagalase testing. Although nagalase is not specific for any particular histological type of cancer, nevertheless, its decrease following GcMAF treatment is considered an index of the therapeutic efficacy of GcMAF since nagalase activity is proportional to tumour burden. Nagalase testing was performed at ELN Laboratories (Bunnik, The Netherlands) following the procedure published by Yamamoto et al. Nagalase activity was determined by using an endpoint enzymatic assay using a chromogenic substrate. ELN Laboratories established a reference range of 0.32 0.95 nM/min/mg of substrate based on serum collected from healthy volunteers, a range slightly higher than that previously reported which was between 0.35 and 0.65 nM/min/mg. Further studies on higher numbers of subjects will establish which reference range is more appropriate. In any case, since all determinations were performed in the same laboratory, a relative decrease of nagalase following GcMAF administration was therefore used as an index of

its therapeutic efficacy. Statistical methods. Statistical comparison between pre-post treatment levels of nagalase was performed by Students t-test. Data in Table 1 are presented as meansS.E.M.. Results GcMAF-activated macrophages appeared as small cells that surrounded human breast cancer cells (Fig. 1). The nucleus of the macrophages is well stained, whereas the chromatin in the nucleus of the cancer cell appears fragmented and disorganized. The nucleoli, however, are still recognizable; this phenomenon can be interpreted as an index of remaining synthetic activity as expected in cells undergoing active apoptosis. The cytoplasm of macrophages appears vacuolated thus suggesting active phagocytosis. GcMAF-activated macrophages emit cytoplasmic extrusions that search for contact with the membrane of cancer cells. Human breast cancer cells show a peculiar aspect; the chromatin in the nucleus appears fragmented and, the cytoplasm appears to be indented as if the macrophages in that region were actively deconstructing the cytoplasmic assembly of the cancer cell. Fig. 1 Fig. 2 Phase contrast microphotography from time-lapse recording of co-culture of GcMAFactivated macrophages and human breast cancer cells, Fig. 2. (A) Day one of co-culture; the cancer cells form an irregular layer. Individual cancer cells can be recognized. GcMAF-activated macrophages appear as small cells that are attached to the cancer cells, in most cases above them. (B) Day seven of co-culture. No individual cancer cell can be recognized. Their apoptotic bodies are grouped together in the center of the field, and most of the field is empty of cancer cells. Most GcMAF-activated macrophages surround and infiltrate the mass of cancer cell debris in the center. Effects of GcMAF on human breast cancer cells, Fig. 3. MCF-7 cells were starved in serum-free medium for 24 h and incubated with GcMAF for further 24 h. At the end of the incubation period, cells were fixed and stained and the plates were photographed under a microscope at low magnification to appreciate the formation of the typical cancer cell clusters. GcMAF was dissolved in a solvent designed to fit its molecular structure; in particular, the solvent was designed to fit the hydrophobic domains binding vitamin D and fatty acids as well as the hydrophilic domain where GalNAc is attached to threonine at position 420. Upper panel: Control. Human breast cancer cells form several clusters Each cluster is formed by about 40 cells. Lower panel: GcMAF (0.2 ng/ml; 4 pM). The dramatic reduction in clusters is clearly evident. Once dissolved in this particular solvent adapted to its molecular structure, GcMAF exerted a powerful anti-cancer effect at extremely low concentration. Effects of GcMAF in human cancer A series of cases describing the results obtained administering GcMAF to patients with diverse types of cancers at advanced stages, often referred to as relatively incurable. The clinical cases reported here are heterogeneous and in all cases, GcMAF treatment was initiated at late stages of tumour progression since it is understandable that conventional therapies were preferred at earlier stages. Thus, most of the cases described here fall in the category of the so called compassionate treatment; in fact, most of them

had undergone conventional anti-cancer treatment in the previous years and they had referred to GcMAF treatment when other conventional treatments had proven ineffective or intolerable. Table 1. Nagalase dataset for pre-post GcMAF therapy. GENDER AGE (years) Male/ Female Pre-last nagalase difference Nagalase level Disease Pre draw date Pre result First-post draw date First-post result Days between pre-first post draw Last draw date Last result Days between pre-last draw 1) M 64 Bladder CA October, 2012 2.90 January, 2013 2.60 92 -0.30 2) M 63 Bladder CA July, 2011 3.10 February, 2012 2.30 215 October, 2012

1.40 458 -1.70 3) F 69 Bladder CA May, 2011 4.10 October, 2011 2.30 153 December, 2012 0.75 580 -3.35 4) F 60 Ovarian CA June, 2012 3.30 June, 2012 3.20 7 November, 2012 2.80 153 -0.50 5) F 62 Ovarian CA August, 2012 2.70 February, 2013 2.00 184 -0.70 6)

F 61 Ovarian CA December, 2012 2.60 February, 2013 2.20 62 -0.40 7) M 67 Prostate CA August, 2012 3.40 December, 2012 2.80 122 -0.60 8) M 76 Prostate CA April, 2011 2.00 August, 2011 1.20 122 December, 2012 0.75 610 -1.25 9) M 65 Prostate CA October, 2011 1.90 February, 2012 1.70

123 October, 2012 1.20 366 -0.70 10) F 66 Breast CA August, 2011 1.70 January, 2012 1.00 153 December, 2012 0.60 487 -1.10 11) F 63 Breast CA May, 2011 5.60 October, 2011 2.90 153 October, 2012 1.10 519 -4.50 12) M 63 Tongue Squamous Cell CA July, 2012 3.00 September, 2012 1.50 62

December, 2012 1.00 153 -2.00 13) F 55 Tongue Squamous Cell CA November, 2012 1.20 February, 2013 0.87 92 -0.33 14) M 54 Colorectal CA July, 2012 3.90 October, 2012 2.00 92 -1.90 Head/Neck Squamous Cell CA June, 2012 2.90 July, 2012 2.70 30 February, 2013 2.00 245 -0.90 October, 2011 2.00 153 December, 2012 0.90 580

-3.80 September, 2012 1.10 92 -0.40 January, 2013 1.20 214 0.20 15) F 58 16) M 72 Larynx CA May, 2011 4.70 17) F 35 Squamous Cell CA June, 2012 1.50 18) F 69 Follicular Lymphoma June, 2012 1.00 19) F 66 Lymphoma August, 2012 2.20 November, 2012 1.90 92 -0.30

42 Anaplastic Oligodendroglioma Grade III November, 2012 3.00 January, 2013 2.60 61 -0.40 610 20) M August, 2012 1.30 61 MAX. 5.60 3.20 215 2.80 min. 1.00 1.00 7 0.60 61 Mean 2.84 2.01* 112 1.59** 263 S.E.M. 0.26 0.22 19.17 0.17 44.90 Narrative description of some notable clinical cases from The Netherlands. The following reports were collected and

communicated by Dr. Steven Hofman, MD, CMC-Capelle a/d Ijssel, The Netherlands. GcMAF (100 ng) was administered by weekly injection following the indications outlined in the literature. The original reports is in italics. The case number refers to the number in Table 1. Female, born 1947. Carcinoma of left breast (found on survey), operated with sentinel nodes in 2010, chemotherapy 4 of 6 series, no specific complaints left. Still some malaise, fatigue and sleepdisorder. Nagalase level at presentation on August 9, 2011: 1.70. January 16, 2012: 1.00. March 12, 2012: 0.72. December 11, 2012: 0.60. GcMAF-treatment (predominantly intravenous route) combined with acupuncture. GcMAF discontinued in April 2012. Aspecific complaints diminished. Patient still seen every few months. A significant decrease of nagalase level can be observed after 5 months of treatment. Such a decrease continued after interruption of GcMAF treatment, reaching normal values about 16 months since the beginning of the treatment. According to the literature, normalization of nagalase level in breast cancer patients is considered an index of eradication of the tumour burden. Female, born 1950. Carcinoma of left breast, specific complaints, metastases probable. After local operation, irradiation of thorax, combined with chemotherapy, Herceptin-therapy. Partly complaints in association with treatments. Nagalase level at presentation on May 11, 2011: 5.60. October 6, 2011: 2.90. February 21, 2012: 1.80. October 18, 2012: 1.10. Treated with intramuscular, later intravenous GcMAF, and a few acupuncturetreatments. No further complaints (subsided in 3-6 weeks), still in intravenous GcMAF-regime. A significant decrease of nagalase level can be observed after 5 months. After about 17 months of GcMAF treatment, nagalase levels are approaching normal values. Conclusions 1 Our results support and reinforce the hypothesis that GcMAF has multiple biological activities that account for its powerful anticancer effects, possibly through molecular interaction with the VDR that in turn is responsible for a multitude of non-genomic as well as genomic effects. Conclusions 2 The clinical response to GcMAF was robust and significant trends emerge evident. All patients, but one, showed significant decrease of nagalase levels following GcMAF weekly injections. Nagalase decrease was associated with improved clinical conditions and no adverse side effects were reported. The observation reported here confirm that GcMAF is a stronghold in the immunotherapy of cancer.

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