Therapeutic efficacy of Gc protein-derived

macrophage activating factor (GcMAF)

for adenocarcinoma.

Nobuto Yamamoto, Masahiro Urade, and Masumi Ueda.

Socrates Institute for Therapeutic Immunology, Philadelphia, PA

and Hyogo College of Medicine, Hyogo, Japan.

Inflammation in Cancerous and Noncancerous Tissues Inflammation Induced by Administration of BCG

a. b.

a. Intratumor administration of BCG results in eradication of

b. Administration of BCG to noncancerous tissues results in no

Refs: 1. Morton, D. et al. 1970, Surgery 68: 158-164.

2. Zbar, B. and Tanaka, T. 1971, Science 172: 271-273.

local as well as metastasized tumors, indicating development of immunity against the tumors.

significant effect on tumors.

O

phospholipase A 2

I. Ester phospholipids:

O

H2 C OC(CH 2 )n CH 3

H3 C(CH 2 )n CO C H

O

H2 C O P O(CH 2 )2 N(CH 2 )3

O

OH

A. O

H2 C OC(CH 2 )n CH3

HO C H

H2 C O P O(CH 2 )2 N(CH 2 )3

O

OH

B.H2 C OH

HO C H

H2 C O P O(CH 2 )2 N(CH 2 )3

O

OH

C.

acyltransferase

lysophospholipase

II. Ether phospholipids:

H2 C OCH 2 (CH 2 )n CH3

H3 C(CH 2 )n CO C H

O

H2 C O P O(CH 2 )2 N(CH 2 )3

O

OH

D.H2 C OCH 2 (CH 2 )n CH 3

HO C H

H2 C O P O(CH 2 )2 N(CH 2 )3

O

OH

E.H2 C OCH 2 (CH 2 )n CH3

HO C H

H2 C O P OH

OH

F.

phospholipase A 2

acyltransferase

lysophospholipase D

acid phosphatase

Metabolic pathways of ester- and ether-phospholipids

H2 C OCH 2 (CH 2 )n CH3

HO C H

H2 C OH

G.

in inflamed tissues

I. Inflammation in noncancerous tissues

1. Phosphatidylcholine (or other Phospholipids)

Phospholipase A 2

Lysophospholipase

3. Inert compounds

1. Alkylphospholipids

Phospholipase A 2

Lysophospholipase D and acid phosphatase

3. Alkylglycerols: are potent macrophage activating agents

Dodecylglycerol ( DDG : one of alkylglycerols)

For macrophage activation, DDG is 400 times more active than lyso-Pc

Conclusion: These information suggests that highly activated macrophages can kill and eradicate cancerous cells.

2. Lysophosphatidylcholine: ( Lyso-Pc ; one of lysophospho-

lipids) is capable of activation

of macrophages.

II. Inflammation in cancerous tissues (e.g., administration of BCG)

2. Lyso-alkylphospholipids: are potent macrophage activating agents

Publications:

1. Yamamoto, N., et al. 1987. Cancer Immuno. Immunother. 25: 185-192.

2. Yamamoto, N. and Ngwenya, B. Z. 1987. Cancer Res. 47: 2008-2013.

3. Yamamoto, N., St. Claire, D. A., Homma, S., and Ngwenya, B. Z. 1988.

Cancer Res. 48:6044-6049.

4. Homma, S. and Yamamoto, N. 1990. Clin. Exp. Immunol. 79: 307-313.

activates macrophages

II. In vitro: Cultivation of [Macrophages] + lyso -Pc or DDG

lyso-Pc or DDG

macrophages alone

no macrophage activation

III. In vitro: Cultivation of [Macarophages + lymphocytes] + lyso -Pc or DDG

lyso-Pc or DDG

IV. Macrophage activation signaling pathway

macrophages + lymphocytes

I. In vivo: Administration of lyso-Pc (20 µg) or DDG (50ng) into mice

lyso-Pc or DDG

B T Mφ activation

activation

B

BT

T

Lyso-Pc or DDG

Proactivating factorB

Mφ

Activated M φ

Mφ

Inflammation primed macrophage activation requires participation of B and T cells.

TB

To search for macromolecular factor to activate macrophages(macrophage activating factor)

1. In serum free medium

lyso-Pc or DDG

Suggesting macrophage activation requires at least one serum component.

2. Macrophage activation requires serum vitamin D binding proteins

lyso-Pc or DDG

+ Gc protein (1 ng/ml)B T Mφ

activation

(known as Gc protein)

B T Mφ no activation

Publications:

1. Yamamoto, N., Homma, S. and Millman, I. 1991. J. Immunol. 147: 273-280.

2. Yamamoto, N. and Homma, S. 1991. Proc. Natl. Acad. Sci. USA 88: 8539-8543.

3. Yamamoto, N., Kumashiro, R., Yamamoto, M., Willett, N. P. and Lindsay, D. D.

1993. Infect. Imm. 61: 5388-5391.

4. Yamamoto, N. and Kumashiro, R. 1993. J. Immunol. 151: 2794-2902.

5. Yamamoto, N., Willett, N. P. and Lindsay, D. D. 1994. Inflammation 18: 311-322.

6. Yamamoto, N., Naraparaju, V. R. and Asbell, S. O.1996. Cancer Res. 56: 2827-

7. Yamamoto, N. 1996. Molecular Immunol. 33: 1157-1164.

8. Yamamoto, N. and Naraparaju, V. R. 1997. Cancer Res. 57: 2187-2192.

9. Koga, Y., Naraparaju, V. R. and Yamamoto, N. 1999. Proc. Soc. Exp. Biol. Med.

220: 20-26.

2931.

12Val

13Cys

14Lys

15Glu

16Phe

17Ser

18His

19Leu

20Gly

21Lys

22Glu

23Asp

24Phe

25Thr

26Ser

27Leu

28Ser

29Leu

30Val

31Leu

32Tyr

33Ser

34Arg

35Lys

36Phe

37Pro

38Ser

39Gly

40Thr

41Phe

42Glu

43Gln

44Val

45Ser

46Gln

47Leu

48Val

49Lys

50Glu

51Val

52Val

53Leu

54Leu

55Thr

56Glu

57

Ala58Cys

59Cys

60Ala

61Glu

62Glu

63Ala 64

Asp65Pro

66Asp

67Cys

68Tyr

69Asp

70Thr

71Arg

72Thr

73Ser

74Ala

75Leu

76Ser

77Ala

78Lys

79Ser

80Cys

81Glu

82Ser

83Asn

84Ser

85Pro

86Phe

87Pro

88

Val

89His

90Pro

91Gly

92Thr

93Ala

94Glu

95Cys

96Cys

97Thr

98Lys

99Glu100

Gly

101Leu 102

Glu103Arg

104Lys

105Leu

106Cys

107Met 108

Ala

109Ala

110Leu

111Lys

112His

113Gln

114Pro

115Gln

116Glu

117Phe

118Pro

119Thr

120Tyr

121Val

122Glu

123Pro

124Thr

125Asn

126Asp127

Glu

128 Ile 129

Cys130Glu

131A l a

132Phe

133Arg

134Lys

135Asp

136Pro

137Lys

138Glu

139Tyr

140Ala

141Asn

142Gln

143Phe

144Met

145Trp

146Glu

147Tyr

148Ser

149

Thr150Asn 151

Tyr

152Glu153

Gln154

Ala155

Pro156Leu

157Ser

158

Leu159

Leu160

Val161

Ser162

Tyr163

Thr164Lys

165Ser

166

Tyr167Leu

168Ser

169

Met170

Val171

Gly172

Ser173

Cys174

Cys175

Thr176

Ser177

Ala

178Ser 179

Pro180Thr

181Val

182Cys

183Phe

184Leu

185Lys

186Glu

187Arg

188Leu

189Gln

190Leu

191Lys

192His

193

Leu194

Ser195

Leu196Leu

197

Thr198

Thr199Leu

200

Ser201

Asn202

Arg203

Val

204Cys

205Ser

206Gln

207Tyr

208Ala

209Ala

210Tyr

211Gly

212Glu

213Lys

214Lys

215Ser

216Arg

217Leu

218Ser

219Asn

220Leu

221Ile

223Leu

222Lys

224Ala

225Gln

226Lys

227Val

228Pro

229Thr

231

Asp232Leu

233

Glu234

Asp235

Val236Leu

237Pro

238Leu

239

Ala240

Glu241

Asp242

Ile243

Thr244Asn

245

Ile246

Leu247

Ser248Lys

249Cys

250

Cys251

Glu252

Ser230Ala

253Ala

254Ser

255

Glu256Asp

257

Cys258

Met259

Ala260

Lys261

Glu262

Leu263

Pro264

Glu265

His266

Thr267

Val268

Lys269

Leu270

Cys271

Asp272

Asn273

Leu274

Ser275

Thr276Lys

277Asp

278Ser

279Lys

280Phe

281

Glu282

Asp283

Cys284

Cys285

Gln286

Glu287

Lys288

Thr289Ala

290Met

291Asp

292

Val293

Phe294Val

295

Cys

296

Thr297Tyr

298Phe 299

Met

300Pro301

Ala302

Ala303

Gln304Leu

305Pro

306

Glu307Leu

308Pro

309Asp

310

Val311Arg

312Leu

313Pro

314Thr

315Asn

316Lys

317Asp

318Val

319Cys

320Asp

321Pro

322Gly

323Asn

324Thr

325Lys

326Val

327Met

328Asp

329Lys

330Tyr

331Thr

332Phe

333

Glu334

Leu335Ser

336Arg

337Arg 338

Thr

339

His340Leu

341Pro

342Glu

343Val

344Phe

345Leu

346Ser

347Lys

348Val

349Leu

350Glu

351Pro

352Thr

353Leu

354Lys

355Ser

356Leu

357Gly

358Glu

359Cys

360Cys

361Asp

362Val363

Glu

364Asp 365

Ser366Thr

367Thr

368Cys

369Phe

370Asn

371

Ala372

Lys373

Gly374Pro

375Leu

376Leu

377Lys

378Lys

379Glu

380Leu

381Ser

382Ser

383Phe

384 Ile

385Asp

386Lys

387Gly

388Gln

389Glu

390Leu

391Cys

392Ala

393Asp

394Tyr

395Ser

396Glu

397Asn

398Thr

399Phe

400Thr

401Glu

402Tyr

403Lys

404Lys

405Lys

406Leu

407Ala

408Glu

409Arg

410Leu

411Lys

412

Ala413Lys

414Leu

415Pro

416Glu

417Ala

418Thr

419Pro

420Thr

421Glu

422Leu

423Ala

424Lys

425Leu

426

Val427

Asn428Lys

429Arg

430Ser

431Asp

432Phe

433Ala

434Ser

435Asn

436Cys

437Cys

438Ser

439 Ile440

Asn

441Ser 442

Pro443Pro

444Leu

445Tyr

446Cys

447Asp

448Ser

449Glu

450

Ile451

Asp452

Ala453Glu

454Leu

455Lys

456Asn

457

Ile458

Leu

1Leu

2Glu

3Arg

4Gly

11Lys

5Arg

6Asp

7Tyr

8Glu

9Lys

10Asp

COOH

NH2

Domain I

Domain II

Domain III

Vitamin D-binding protein (known as Gc protein)

Gc protein Macrophage activatingfactor (MAF)

....

Gal

SA

GalNAc Thr

....

SA

GalNAc Thr

....

GalNAc Thr

β-galactosidaseof B cells *

▼

sialidaseof T cells

▼

Gc protein Macrophage activatingfactor (GcMAF)

....

Gal

SA

GalNAc Thr

....

SA

GalNAc Thr

....

GalNAc Thr

β-galactosidaseimmobilized immobilized

sialidase

▼

In vitro Tumoricidal Capacity of Macrophages Activated by GcMAFa. Time course observation of tumoricidal process with trypan blue penetration.

b. Tumoricidal capacity of GcMAF-treated human macrophages for prostate cancer cells LNCaP (i) and breast cancer cells MDA-MB-231 (ii)

100

50

4 18

Hrs of cocultivation

100

50

4 18

Hrs of cocultivation

, tumor cells only; , tumor cells with macrophages; , tumor cells with activated macrophages

Approximately 3.5 x 10 5 tumor cells were cultured with 1.1 x 10 6 macrophages/well.

(i) (ii)

Mφ

Cancer

Macrophages were activated by preincubation with GcMAF (100pg/ml) for 3hr.

Characterization of monocytes/macrophages, lymphocytes andSerum Gc protein of individual oral cancer patients.

nmole superoxide produced/min/10 6 phagocytesPatient

Assayed on: phagocytes* lymphocytes/phagocytes**

12

3

4

5

6

78

9

10

Healthy humans

0.210.66

0.71

0.40

0.62

0.56

0.330.72

0.29

0.620.27

6.385.99

6.34

5.96

6.83

5.92

5.845.96

6.70

6.346.68

5.876.03

5.65

5.72

5.64

5.87

5.945.63

6.26

6.086.22

2.611.42

5.19

1.07

3.45

1.11

0.721.19

2.14

0.314.25

* lyso-Pc untreated. ** lyso-Pc-treated lymphocytes + phagocytes.

Precursor activity of serum Gc protein were analyzed using 0.1% patient serum.

none 100pg GcMAF 1ng Gc protein 0.1% serumNo.

Deglycosylated Gc proteinGc protein

Gc protein Macrophage activating

factor (MAF)

....

Gal

SA

GalNAc Thr

....SA

GalNAc Thr

....

GalNAc Thr

....

Thr

....

Gal

SA

GalNAc Thr

sialidaseof T cells

▼

β-galactosidaseof B cells*

▼

α-N-acetylgalacto-

saminidase

▼

....

........

....

....

25

20

15

10

5

00 200 400 600 800 1000 1200

Tumor weight (mg)

Correlation between serum α-N-acetylgalactosaminidase

activity and tumor burden (measured by total weight) in

nude mouse transplanted with human oral squamous cell

carcinoma (KB) cell line.

0 2 4 8 10 12 14 16 18 20 22 246

Weeks

GcMAF therapy for prostate cancer patients

0

1

2

3

4

5

6123456789

10111213141516

Healthy Control

Weekly administration of 100ng GcMAF

0 2 4 8 10 12 14 16 18 20 2260

1

2

3

4

5

6

7

Weeks

GcMAF therapy for breast cancer patients

123456789

10111213141516

Healthy Control

Weekly administration of 100ng GcMAF

Tumoricidal capacity of GcMAF-treated human macrophagescancer cells LNCaP (a) and breast cancer cells for prostate

MDA-MB-231 (b).

, tumor cells only;, tumor cells with macrophages;, tumor cells with activated macrophages;, patient serum coated cancer cells with activated macrophages.

In vitro Tumoricidal Capacity of Macrophages Activated by GcMAF

100

50

4 18

(a)

hrs of cocultivation

100

hrs of cocultivation

(b)

50

4 18

Approximately 3.5 x 10 5 tumor cells were cultured with

Time course observation of 1.1 x 10 6 macrophages/well.

tumoricidal process was performed by trypan blue

exclusion vital assay.

IMMUNE DEVELOPMENTPrincipal Immune Development Cascade

Inflammation

Inflamed lesions release lysophospholipids(chemotactic agents)

LymphocytesMacrophages

(activation)

Activated macrophages(Highly activated

Cell killing

macrophages)phagocytosis

(degradation product/

antigen processing

antigen presentation

B and T lymphocytes

Development of Humoral and Cellular Immunity*

*Macrophage activation is the first mandatory step for immune development. Thus, lack of macrophage

activation leads to immunosuppression.

fragment component)