Use of Endotoxin in Cancer Immunotherapy and ... - ACS Publications

Use of Endotoxin in Cancer Immunotherapy

10

a n d C h a r a c t e r i z a t i o n of Its N o n t o x i c b u t A c t i v e Lipid

A

Components

KUNI TAKAYAMA and NILOFER QURESHI—William S. Middleton Memorial Veterans Hospital, Madison, WI 53705 EDGAR RIBI and JOHN L. CANTRELL—Ribi ImmunoChem Research, Inc., Hamilton, MT 59840

Downloaded by AUBURN UNIV on September 12, 2017 | http://pubs.acs.org Publication Date: September 29, 1983 | doi: 10.1021/bk-1983-0231.ch010

1

KENICHI AMANO —United States Public Health Service, National Institutes of Health, National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratory, Hamilton, MT 59840

When crude endotoxin from the heptose-less mutant of Salmonella typhimurium is combined with trehalose dimycolate from mycobacteria in o i l droplets and injected directly into estab lished tumors (line 10 hepatocellular carcinoma) in syngeneic guinea pigs, rapid regression of the tumors occurs and over 90% of the animals are cured. The three required components for activity in this tumor model are: (a) the endotoxin; (b) the mycobacterial adjuvant, trehalose dimycolate; and (c) a compound satisfying the minimal structural requirement (muramyl dipeptide) for adjuvant activity by bacterial c e l l wall materials. The mycobacterial c e l l wall skeleton is able to replace the latter two components. Since endotoxin is very toxic to humans, a method was sought to render it nontoxic and yet retain its tumor regression a c t i v i t y . We detoxified endotoxic extracts and diphosphoryl l i p i d A obtained from S. typhimurium G30/C21 by controlled acid hydrolysis to yield a product that retained the ability to synergistically enhance the antitumor properties of mycobacterial adjuvants. The chemical structure of the nontoxic product was established by f i r s t purifying i t by preparative thin layer chromatography, anal yzing the purified l i p i d A by reverse-phase high performance liquid chromatography, and finally by determining its exact mole cular mass by fast atom bombardment mass spectrometry. The non toxic but active product was shown to be a 4'-monophosphoryl lipid A containing ester-linked lauroyl-, 3-hydroxymyristoyl-, and 3-myristoxymyristoyl grouips. This l i p i d may represent a potential candidate for use in the immunotherapy of human cancer. Early History of Tumor Immunology The suggestion that the control of cancer may be effected by immunologic methods was made about 100 years ago. At that time, William Coley observed that tumors either partially or totally 1

Current address: Hirosaki University School of Medicine, Department of Bacteriology, Hirosaki 036, Japan 0097-6156/83/0231-0219$06.00/0 © 1983 American Chemical Society

Anderson and Unger; Bacterial Lipopolysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

BACTERIAL LIPOPOLYSACCHARIDES

220

regressed i n p a t i e n t s following an acute b a c t e r i a l i n f e c t i o n ( 1 ) . Today we know that an aroused immune system, which plays a protec t i v e r o l e against i n f e c t i o u s disease, also plays a major r o l e i n the host's defense against cancer. The e f f e c t i v e ingredient of C o l e y s b a c t e r i a l vaccine appeared to be endotoxin, which caused hemorrhagic n e c r o s i s of the tumor. Although the antitumor a c t i v i t y of endotoxin has been e x t e n s i v e l y studied for at l e a s t 50 years, endotoxin had come to be regarded as of questionable value for the treatment of tumors. I t r a r e l y led to tumor e l i m i n a t i o n with the concomitant production of systemic tumor immunity but rather to p a r t i a l r e g r e s s i o n followed by resumption of growth. I t was not u n t i l the l a t e 1960's that encouraging experimental and c l i n i c a l r e s u l t s were obtained from the treatment of tumors with b a c t e r i a l preparations other than endotoxins, p r i m a r i l y with an attenuated a n t i t u b e r c u l o s i s vaccine c o n s i s t i n g of l i v i n g bac t e r i a of Mycobacterium bovis s t r a i n BCG alone or admixed with tumor c e l l s . However, the use of v i a b l e c e l l s of BCG caused complications• Because of the need for more potent, nonviable immunotherapeutic agents that can be administered without harmful side e f f e c t s , a major o b j e c t i v e of our research e f f o r t was to i d e n t i f y , i s o l a t e , and evaluate chemically defined m i c r o b i a l components that are e f f e c t i v e antitumor agents.


f

Development of Well-Defined C e l l - F r e e Components Preparations of BCG c e l l w a l l i n o i l droplets were as e f f e c t i v e as v i a b l e BCG i n inducing r e g r e s s i o n of e s t a b l i s h e d s k i n tumors ( l i n e 10) of inbred guinea pigs and i n e l i m i n a t i n g lymph node metastases upon i n t r a l e s i o n a l i n j e c t i o n (2). By f r a c t i o n a t i n g the BCG c e l l wall we have obtained components that r e t a i n antitumor a c t i v i t y , and have the p o t e n t i a l advantages of increased potency and reduced a l l e r g e n i c i t y ( 3 , 0 • To i s o l a t e and define the c e l l wall components required for tumor r e g r e s s i o n , we f i r s t digested the c e l l walls with p r o t e o l y t i c enzymes to remove proteins and then exhaustively extracted them with organic solvents to l i b e r a t e free l i p i d s . The r e s u l t i n g c e l l wall skele ton (CWS), an i n s o l u b l e , polymeric mycolic acid-arabinogalactanmucopeptide, had reduced antitumor a c t i v i t y compared with c e l l w a l l s . But f u l l a c t i v i t y was restored when CWS was combined with trehalose dimycolate ( P 3 ) , obtained from the free l i p i d s of the c e l l w a l l (5). E f f i c a c y of Endotoxin i n Tumor Immunology We found that P 3 combined with c e l l walls of endotoxin con t a i n i n g gram-negative b a c t e r i a , such as E s c h e r i c h i a c o l i and Salmonella, induced complete r e g r e s s i o n of l i n e 10 tumors ( 6 ) . This led to a r e i n v e s t i g a t i o n of the value of endotoxins, which were known to be equally powerful m i c r o b i a l adjuvants (poten-


10.

TAKAYAMA ET AL.


221


t i a t o r s of immune responses) as the above described mycobacterial c e l l w a l l f r a c t i o n s , namely CWS and P3. In agreement with the e a r l y f i n d i n g s , when the endotoxin was i n j e c t e d alone into l i n e 10 tumors, we observed a n e c r o t i c reac t i o n leading to p a r t i a l r e g r e s s i o n and the tumor s h o r t l y resumed i t s growth. However, when c e r t a i n preparations of endotoxin, i n combination with P3 i n o i l d r o p l e t s , were i n j e c t e d into e s t a b l i s h e d tumors, a high r a t e of cures concomitant with the development of s p e c i f i c tumor immunity was a t t a i n e d . The animals r e j e c t e d a subsequent challenge with a l e t h a l dose of l i n e 10 tumor c e l l s ( 6 ) . The data are summarized i n Table I . A l l of the tumors disappeared when 150 yg of endotoxin obtained from the rough mutant of Salmonella typhimurium was combined with 50 yg of Table I . S y n e r g i s t i c E f f e c t Between C e l l Wall Skeleton (CWS) and Endotoxins (ET) i n Regressing Line 10 Tumors M a t e r i a l a s s o c i a t e d with o i l d r o p l e t s i n j e c t e d i n t o tumors ET P3 ET + P3 P u r i f i e d ET + P3 CWS CWS CWS + p u r i f i e d ET P u r i f i e d ET + P3 + ACP 150 ACP + P 3

a

b

C

a

c

R i b i et a l . (7) Takayama ejt a l . (9)

b

Cured/Total Dose (yg) 0/8 150 0/217 150 8/8 150 + 50 0/8 150 + 50 14/16 300 1/16 50 8/8 50 + 50 54/67 + 50 + 150 0/9 150 + 50

Percent cured 0 0 100 0 88 6 100 81 0

R i b i et a l . (8)

P3. When the endotoxin preparation was freed of peptides, phospholipids, and d i v a l e n t c a t i o n s , i t lacked the tumor r e g r e s s i v e potency. We noted muramic a c i d , a l a n i n e , and glutamic a c i d among the p r i n c i p a l nitrogenous components present i n the endotoxic e x t r a c t , and observed that the p r o p o r t i o n of each of these components was lowered s i g n i f i c a n t l y during the preparation of p u r i f i e d endotoxin (10). These are the components that make up the minimal s t r u c t u r a l e n t i t y , N-acetylmuramyl-L-alanyl-Disoglutamine (muramyl d i p e p t i d e ) , of the mucopeptide moiety of the b a c t e r i a l CWS that i s responsible f o r adjuvant a c t i v i t y (11). We considered the p o s s i b i l i t y that the product of a u t o l y s i s of the Salmonella CWS may have been co-extracted with the endotoxic glycolipids. Since the CWS of mycobacteria but not that of Salmonella contains mycolic acid esters ( t o replace P3 i n a d d i t i o n to the peptidoglycan moiety, i t was not s u r p r i s i n g to f i n d that the tumor r e g r e s s i v e potency of the p e p t i d e - f r e e endotoxin could be restored by the a d d i t i o n of mycobacterial CWS (10). As l i t t l e as 50 yg of CWS and 50 yg of p u r i f i e d endotoxin s u f f i c e d to give 100% r e g r e s s i o n . A t e n - f o l d lower quantity of p u r i f i e d endotoxin



222

s t i l l led to a cure rate of 78%. The tumor r e g r e s s i v e potency of p u r i f i e d endotoxin also could be restored by the a d d i t i o n , to the p u r i f i e d endotoxin plus P3, of a preparation of acetone-chloroform p r e c i p i t a t e (ACP), a nontoxic peptide-containing side f r a c t i o n obtained during the i s o l a t i o n of endotoxin (10). The tumors treated with t h i s endotoxin mixture regressed r a p i d l y ( i n about one week), whereas tumors treated with a 300 yg dose of CWS plus P3 regressed slowly ( i n about 2-3 weeks).


The Problem of T o x i c i t y of Endotoxin Human, horse, dog, and r a b b i t are known to be h i g h l y suscep t i b l e to the toxic e f f e c t s of endotoxin whereas small animals such as guinea pig and mouse are r e l a t i v e l y r e s i s t a n t (Table I I ) . As an example, a s i n g l e i n j e c t i o n of 150 yg of endotoxin can be l e t h a l to a horse. Because of t h i s high s u s c e p t i b i l i t y of humans to endotoxin, i t s p o t e n t i a l s f o r c l i n i c a l a p p l i c a t i o n have not been r e a l i z e d . In the past, endotoxin preparations were u s u a l l y described by t h e i r degree of t o x i c i t y and p y r o g e n i c i t y . We and other i n v e s t i g a t o r s had p r e v i o u s l y explored the p o s s i b i l i t y of using chemical m o d i f i c a t i o n techniques to s e l e c t i v e l y reduce the t o x i c i t y and p y r o g e n i c i t y of endotoxins while r e t a i n i n g adjuvant i c i t y (15-19). The aim was to provide chemically defined, non t o x i c adjuvants which were capable of enhancing n o n s p e c i f i c r e s i s tance to b a c t e r i a l i n f e c t i o n s and of s y n e r g i s t i c a l l y enhancing the tumor r e g r e s s i v e potency of mycobacterial CWS (10). Because the experiments were done with heterogenous mixtures of endotoxin whose s t r u c t u r e s are unknown, the observed biological activity could not be r e l a t e d d i r e c t l y to any s p e c i f i c component(s). An Approach to P u r i f y i n g Endotoxin and I t s D e r i v a t i v e s I t was

our working hypothesis that complete

s o l u b i l i z a t i o n and

Table I I . L e t h a l Dose of Endotoxin and Detox Detox Detox i s less toxic by f a c t o r of Test subject (ug) >20->100 Mouse >10,000 — Guinea p i g ND Horse >130 >20,000 Dog >40 >4,000 — Human^ ND >1,500->15,000 Rabbit >15,000 Chick embryoS >10,000 >10 C a n t r e l l and R i b i , unpublished r e s u l t s ^ M i l n e r et a l . (12) R i b i jit a U (10) Bottoms j 2 t jrt. (13) Hinshaw et a l . T l 4 ) f S u s c e p t i b i l i t y i s estimated to be s i m i l a r to that of dog and rabbit. &Takayama et^ ail. (9) 3

Endotoxin (U8) 100-500 >300 150 100 1-100 1-10 10 yg) and nonpyrogenic (FI40J 20 yg) (Table I V ) . We have designated t h i s product nontoxic l i p i d A (Detox) and demonstrated i t s low t o x i c i t y i n four animal species (Table I I ) . The nontoxic l i p i d A i n combination with P3 and ACP (or CWS) r e t a i n e d a degree of tumor r e g r e s s i v e potency (80% cures) s i m i l a r to that observed with the toxic l i p i d A (88% cures) and the p u r i f i e d endoxtoxin (81% c u r e s ) . R e s u l t s of chemical a n a l y s i s showed that the glucosamine and t o t a l f a t t y acid contents of the KDO depleted, toxic l i p i d A and the nontoxic l i p i d A were e s s e n t i a l l y the same but that the non t o x i c l i p i d A was s i g n i f i c a n t l y lower i n the phosphorus content (Table V ) . The molar r a t i o of glucosamine:phosphorus:fatty acids was 2:2:4 f o r the toxic l i p i d A and 2:1:4 f o r the nontoxic l i p i d A. The r e l a t i v e molar d i s t r i b u t i o n of normal f a t t y acids ( l a u r i c , m y r i s t i c , and p a l m i t i c a c i d s ) and the 3-hydroxymyristic acid did not i n d i c a t e a c o r r e l a t i o n between the content of these components and t o x i c i t y . The nontoxic l i p i d A possessed as high a tumor r e g r e s s i o n a c t i v i t y when combined with CWS as did the p u r i f i e d



224

endotoxin from which i t was prepared (Table V l ) 0 , C a n t r e l l and R i b i , unpublished r e s u l t s ) . Whereas a 150 y g dose of endotoxin was l e t h a l to a horse (Table I I ) , the a d m i n i s t r a t i o n of 20,000 yg of the nontoxic l i p i d A caused no detectable l e t h a r g i c r e a c t i o n s or r i s e i n the body temperature. Table IV. Antitumor E f f e c t o f Toxic and Nontoxic Pyrogenicity for r a b b i t s

Material tested

CELD ,ug 50


a

Purified ET Toxic l i p i d A Nontoxic l i p i d A (Detox) ACP a

O.026 O.031 >10

(FI40,

yg)

O.049 ND 20

Glycolipids

regression L i n e 10 tumor i +ACP +P3 +P3 cured «, cured % total total 54/67 81 1/26 4 7/8 88 ND 27/32 84 ND

3

b

a


>10 b

0/26

>100

0

—

Takayama et a l . (9)

Table V. Chemical Composition of Endotoxin (ET), and L i p i d A

P u r i f i e d ET,

Total fatty acid Glucosamine Phosphorus KDO nmol/mg (mole/2 moles glucosamine) ET 638 (2) 941 (2.95) 911 (2.86) 1715 (5.37) Purified ET 932 (2) 1055 (2.26) 1134 (2.43) 1821 (3.91) Toxic l i p i d A 1182 (2) 1122 (1.90) 14.1 (O.02) 2658 (4.49) Nontoxic l i p i d A 1157 (2) 713 (1.23) 9.8 (O.02) 2510 (4.30) (Detox)

Material tested 3

b

b

b

Amano £t a l . (21)

3

b


Table V I . Synergy Between Endotoxin (ET), Nontoxic L i p i d A (Detox), and Mycobacterial C e l l Wall Skeleton (CWS) i n Regressing L i n e 10 Tumors 3

M a t e r i a l a s s o c i a t e d with o i l droplets injected i n t o tumors CWS ET CWS + ET

Dose (yg) 50 150 50 + 50 50 + 5 Detox 50 CWS + Detox 50 + 50 25 + 25 50 + 8 Cantre11 and R i b i , unpublished data

Cured/Total 10/48 0/8 8/8 6/8 0/18 50/52 9/10 4/8

l


Percent cured 20 0 100 75 0 96 90 50

10.

TAKAYAMA ET AL.

Use of Endotoxin

in Cancer

Immunotherapy

225

We have shown that the treatment of disaggregated, p u r i f i e d endotoxin with HCl r e p r o d u c i b l y led to complete d e t o x i f i c a t i o n (CELD50, >10 y g ) , whereas s i m i l a r treatment of conventional endo t o x i n e x t r a c t s (with CELD50 of O.004 yg) led to only p a r t i a l d e t o x i f i c a t i o n (CELD50, O.25 y g ) . The f a i l u r e to d e t o x i f y t h i s l a t t e r extract might be due to the aggregation of the endotoxin and i t s p r o t e c t i v e e f f e c t on a c i d - l a b i l e groups, r e s u l t i n g i n incomplete h y d r o l y s i s . T h i s may e x p l a i n the e a r l i e r reported r e s u l t s where a mere 10-fold decrease i n t o x i c i t y was observed f o l l o w i n g a c i d treatment of endotoxic phenol-water e x t r a c t s (22).


P u r i f i c a t i o n of L i p i d A We showed the existence of a t o x i c and a nontoxic l i p i d A f r a c t i o n i n the acid hydrolyzed endotoxin p r e p a r a t i o n (23). These two f r a c t i o n s were separated and the composition was determined on the p u r i f i e d components so that we could r e l a t e s p e c i f i c s t r u c t u r a l features of l i p i d A to both t o x i c i t y and tumor r e g r e s s i o n a c t i v i t y (23). F i g u r e 1 shows the scheme for the p r e p a r a t i o n of p u r i f i e d l i p i d A from endotoxin. j>. typhimurium G30/C21 was extracted by the method of Galanos et a l . (24) and submitted to one of two d i f f e r e n t c o n d i t i o n s of h y d r o l y s i s : (a) O.1 N HCl [ i n methanol-water (1:1, v / v ) ] , 100 °C., 45 min, to y i e l d the crude monophosphoryl l i p i d A ( n o n t o x i c ) , and (b) O.02 M sodium acetate, pH 4.5, 100 °C for 30 min (two c y c l e s ) to y i e l d the crude diphosphoryl l i p i d A (toxic). The O.1 N HCl h y d r o l y s i s product was f r a c t i o n a t e d on a Sephadex LH-20 column (23). Each of these f r a c t i o n s was then separated by p r e p a r a t i v e t h i n layer chromatography (TLC) on s i l i c a gel H (500 ym), with the solvent system chloroform-methanol-waterconcentrated ammonium hydroxide (50:25:4:2, v/v) as p r e v i o u s l y described (23) to y i e l d TLC f r a c t i o n s 1-7 and 1-9 r e s p e c t i v e l y . Chemical A n a l y s i s of P u r i f i e d L i p i d A The r e s u l t s of the chemical a n a l y s i s of these f r a c t i o n s are given i n Table V I I . For the monophosphoryl l i p i d A (TLC-1, -3, and - 5 ) , the glucosamine:phosphate r a t i o was 1.98 to 2.15, whereas Table V I I .

Content

Chemical Analyses of Mono- and Diphosphoryl L i p i d A

(ymol/mg)

Phosphate Glucosamine KDO Glucosamine/Phosphate (molar r a t i o ) a

Qureshi jit a l . (23)

TLC-1 O.54 1.07 O.0 1.98 b

Lipid A Diphosphoryl Monophosphoryl a TLC-5 TLC-3 O.81 O.58 O.52 O.76 1.24 1.12 O.01 O.0 O.0 O.95 2.14 2.15

0

T h i s sample contained 20-25% s i l i c a g e l .



226

S.

TYPHIMURIUM G30/C21


SOLVENT

EXTRACTION

GALANOS-TYPE ENDOTOXIN

1.

O.1

2.

SEPHADEX L H - 2 0 COLUMN

N HCL

CRUDE MONOPHOSPHORYL

HYDROLYSIS

PH 4 . 5

CRUDE

HYDROLYSIS

DIPHOSPHORYL

LIPID A

LIPID A

TLC

TLC BAND #1-7

TLC

TLC BAND #1-9

Figure 1. Scheme for preparing and purifying mono- and diphosphoryl lipid A from endotoxin obtainedfrom S. typhimurium G30/G21.


Anderson and Unger; Bacterial Lipopolysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1983. l4

J4

MINUTES

Conditions: C - Bondedcartridge. 8 mm * 10 cm (Radial pak A, Waters Associates, Inc.) was used with a mobile phase of a linear gradient ofD-IOOty isopropylalcohol-water(9:1, v/v)(solvent B)in acetonitrile-water(9:1, v/v)(solvent A) over a period of60 min at aflow rate of3mL/min. Both solvents contained 5 mM tetrahutylammonium phosphate. For the determination of the radioactivity profile, fractions were collected at 3(i-s inten'als and assayedfor radioactivity.

Figure 2. Reverse-phase HPLC of purified [ C]monophosphoryI lipid A. A, TLC-3; B, TLC-5. The sample sizes were IA and2mg, respectively (60,000 cpm each). The labeled lipid As were obtainedfrom S. typhimurium G30/G21 grown in the presence of[l C]acetate as previously described (23).

MINUTES



228

i n the u n f r a c t i o n a t e d but T L C - p u r i f i e d diphosphoryl l i p i d A, the r a t i o was O.95. A l l f r a c t i o n s were v i r t u a l l y devoid of KDO.


High Performance L i q u i d Chromatography (HPLC) of Monophosphoryl Lipid A The a n a l y s i s of the p u r i f i e d monophosphoryl l i p i d A by reverse-phase HPLC revealed the degree of p u r i t y of the f r a c t i o n s TLC-1, -3, -5, and -7 (23). TLC-1 and -3 each gave one major peak (85%), and these peaks had i d e n t i c a l e l u t i o n times. TLC-5 showed one major (72%) and one minor (18%) peak. TLC-7 resolved into one major (48%) and two minor (21 and 16%) peaks. One of the minor peaks i n TLC-7 (16%) was the overlapping contaminant of the minor components i n TLC-5. Representative r e s u l t s of the HPLC a n a l y s i s of 14 - l a b e l e d TLC-3 and -5 are shown i n Figure 2. C

Fast Atom Bombardment (FAB) Mass S p e c t r a l A n a l y s i s P u r i f i e d monophosphoryl l i p i d A (TLC-1 through -9) were a n a l yzed by FAB mass spectrometry i n the negative mode, gave the r e s u l t s summarized i n Table V I I I . TLC-1 gave a major molecular ion (M-H)" at m/z 1730. I t s e s t e r - l i n k e d f a t t y acid composition would be one mole each of 3-hydroxymyristic, l a u r i c , and 3-myristoxymyristic acids per mole of l i p i d A. TLC-1 also con tained a methyl group ( p o s s i b l y as a methyl gycoside or a methyl phosphate) that was probably added to the l i p i d during acid hydro l y s i s of the sugar 1-phosphate i n the presence of methanol. TLC-3 gave a molecular ion at m/z 1716. I t s e s t e r - l i n k e d f a t t y acid composition was i d e n t i c a l to that of TLC-1. The major component of TLC-5 gave a molecular ion at m/z 1506. I t s e s t e r - l i n k e d f a t t y a c i d composition would be two moles of 3-hydroxymyristic and one mole of l a u r i c acids per mole of l i p i d A. Other minor ions were observed i n TLC-1 through -7. F i n a l l y , TLC-9 gave a molecular i o n at m/z 1098. A molecule of t h i s l i p i d would contain a s i n g l e e s t e r - l i n k e d 3-hydroxymyristic acid r e s i d u e . The e s t e r - l i n k e d f a t t y acid d i s t r i b u t i o n , molecular formulas of the free a c i d s , and t h e i r M were determined as shown i n Table IX. r

Table V I I I .

FAB Mass S p e c t r a l A n a l y s i s of P u r i f i e d Monophosphoryl Lipid A a

m/z TLC F r a c t i o n TLC-1 TLC-3 TLC-5 TLC-7 TLC-9

Molecular i o n 1730 1716 1506 1280 1098

(M-H)" Other ions 1716, 1520, 1294 1520, 1506, 1294, 1534, 1520, 1280 1324, 1308, 1294 None

Reprinted from Ref. 23.


1280

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Immunotherapy

229

Table IX. E s t e r - l i n k e d F a t t y Acid Content, Molecular Formulas and Molecular Weights of F r a c t i o n s of P u r i f i e d Monophosphoryl L i p i d A

a

Monophosphoryl l i p i d A fraction Ester-linked fatty a c i d Molecular Mc (major component) formula MM 0H-C C TLC 1 C95H179U22N2P 1731.2 1 1 1 p

0

14

TLC-3

1

1

1

TLC-5

2

1

0

TLC-7 TLC-9

1 1

1 0

0 0

a


r

1 2

D

c

Qureshi et a U (23) OH-C14, 3-hydroxymyristic acid; MM, C a l c u l a t e d as the free a c i d

C 4H 7022N2P 9

17

H

C80 15lO22N2P N

C66Hl25°19 2

P

C54H103OI8N2P

3-myristoxymyristic

R e l a t i n g the Mono- and Diphosphoryl L i p i d

1717.2 1507.0 1280.8 1098.7

acid

A's

The TLC p u r i f i e d diphosphoryl l i p i d A was hydrolyzed i n O.1 N HCl at 100 °C for 30 min to y i e l d the corresponding monophosphoryl l i p i d A d e r i v a t i v e s as p r e v i o u s l y described (23). These products were then compared with p r e v i o u s l y c h a r a c t e r i z e d monophosphoryl l i p i d A f r a c t i o n s by TLC using s i l i c a gel H (250 ym) and the pre v i o u s l y mentioned solvent system. The TLC f r a c t i o n s -3, -5, -7 of the a c i d hydrolyzed diphosphoryl l i p i d A s e r i e s corresponded with a s i m i l a r l y numbered s e r i e s of monophosphoryl l i p i d A f r a c t i o n s (TLC-3, -5, and - 7 ) . There appeared to be some breakdown of the monophosphoryl l i p i d A products to the lower homologues presumably by the a c i d c a t a l y z e d h y d r o l y s i s of some f a t t y e s t e r l i n k a g e s . TLC-1 and -9 were not analyzed due to small sample s i z e s . The TLC p u r i f i e d diphosphoryl l i p i d A f r a c t i o n s (TLC -3, -5, and -7) were analyzed by FAB mass spectrometry i n the negative mode as p r e v i o u s l y described (23) and the r e s u l t s are shown i n Table X. TLC-3 gave a molecular ion (M-H)~ at m/z 1796; TLC-5, m/z 1586; TLC-7, m/z 1360. As expected, these values were 80 amu or (PO3H2-H) l a r g e r than those for the corresponding monophosphoryl l i p i d A's of the s e r i e s as shown i n Table V I I I . These r e s u l t s e s t a b l i s h e d the s t r u c t u r a l r e l a t i o n s h i p between the mono- and diphosphoryl l i p i d A's. T o x i c i t y and Tumor Regression A c t i v i t y of P u r i f i e d L i p i d A The r e s u l t s of the biological t e s t s c a r r i e d out on the p u r i f i e d monophosphoryl l i p i d A f r a c t i o n s are shown i n Table X I . The chick embryo l e t h a l i t y t e s t showed that both TLC-1 and -3 were nontoxic, whereas TLC-5 e x h i b i t e d some t o x i c i t y . These r e s u l t s i n d i c a t e that there might be two l e v e l s of t o x i c i t y based on s t r u c t u r e . The presence or absence of the sugar 1-phosphate group (and p o s s i b l y some other unknown group) would c o n t r o l the upper



230 Table X.

FAB Mass S p e c t r a l A n a l y s i s and Molecular Determination of the S e r i e s of P u r i f i e d Diphosphoryl L i p i d A F r a c t i o n s

TLC f r a c t i o n TLC-3 TLC-5 TLC-7 C a l c u l a t e d as


a

Molecular ion m/z (M-H)" 1796 1586 1360 the

Weight

M* r

1797.2 1587.0 1360.8

free a c i d

Table XI. T o x i c i t y and Tumor Regression A c t i v i t y of P u r i f i e d L i p i d A Obtained from S. typhimurium G30/C21

CELD50, yg

L i n e 10 tumor r e g r e s s i o n i n guinea pigs Cured/total Percent cured

Material tested Diphosphoryl 92 lipid A 33/36 O.033 Monophosphoryl lipid A 88 7/8 TLC-1 >10 75 6/8 TLC-3 >10 100 8/8 TLC-5 O.199 C a l c u l a t e d sum and average of the diphosphoryl l i p i d A f r a c t i o n s V-VII from the DEAE-cellulose column as reported by Takayama jrt a l . (9; Data from Qureshi j 2 t a l . (23) a

b

a

D

l e v e l of t o x i c i t y ( m a c r o t o x i c i t y ) and the degree or kind of 0a c y l a t i o n could c o n t r o l the lower l e v e l s of t o x i c i t y ( m i c r o t o x i c i t y ) . A l l three of the p u r i f i e d monophosphoryl l i p i d A f r a c t i o n s were a c t i v e i n the tumor r e g r e s s i o n assay. I t i s to be noted that the diphosphoryl l i p i d A mixture i s both toxic and has tumor r e g r e s s i o n a c t i v i t y . We have thus associated the lack of t o x i c i t y and tumor r e g r e s s i o n a c t i v i t y to p r e c i s e chemical s t r u c tures of l i p i d A. S t r u c t u r e of Nontoxic Monophosphoryl L i p i d A The suggested s t r u c t u r e for the nontoxic monophosphoryl l i p i d A i s given i n Figure 3. The phosphate group would occupy the 4'p o s i t i o n of the 2-deoxy-2-amino-g-D-glucopyranosyl-(l->6)-2-deoxy2-amino-D-glucopyranose d i s a c c h a r i d e . A l a u r o y l , 3-myristoxym y r i s t o y l group or a hydrogen (R ) would occupy the 3- and 4p o s i t i o n s and a 3-hydroxymyristoyl residue (R^) would occupy the 6 ' - p o s i t i o n of the d i s a c c h a r i d e . I f there i s a free hydroxyl group at p o s i t i o n 3 or 4, the l a u r o y l group would be associated with one of the two n i t r o g e n - l i n k e d hydroxy f a t t y acids (R^)• 2



10.

TAKAYAMA ET AL.


Figure 3. Structure of nontoxic monophosphoryl lipid A. R, = 3-hydroxymyristoyl; R - lauroyl, 3-myristoxymyristoyl, or H. 2


231


232

This i s the proposed s t r u c t u r e of the h i g h l y O-acylated and non t o x i c TLC f r a c t i o n 3. S i g n i f i c a n c e o f Nontoxic L i p i d A T h i s nontoxic l i p i d may represent a p o t e n t i a l candidate f o r use i n the immunotherapy of human cancer. In a d d i t i o n , i t w i l l be i n t e r e s t i n g to determine to what extent t h i s nontoxic l i p i d A can replace the toxic components i n e l i c i t i n g the numerous other biological a c t i v i t i e s of l i p i d A.


Acknowledgment T h i s research was supported by the Veterans A d m i n i s t r a t i o n .

Literature

Cited

1. Coley, W.B. Ann. Surg. 1891, 14, 199. 2. Zbar, B.; Ribi, E.; Rapp, H.J. Nat. Cancer Inst. Monogr. 1973, 39, 3. 3. Azuma, I.; Ribi, E.; Meyer, T.J.; and Zbar, B. J. Nat. Cancer Inst. 1974, 52, 95. 4. Ribi, E.; McLaughlin, C.A.; Cantrell, J.L.; Brehmer, W.; Azuma, I.; Yamamura, Y.; Strain, S.M.; Hwang, K.M.; Toubiana, R. "Immunotherapy of Human Cancer"; edited by staff of Univ. of Texas System Cancer Center, Houston; Raven Press: New York, 1978, pp. 131-154. 5. Meyer, R.I.; Ribi, E.; Azuma, I.; and Zbar, B. J. Nat. Cancer Inst. 1974, 52, 103. 6. Ribi, E.; Granger, D.L.; Milner, K.C.; and Strain, S.M. J. Nat. Cancer Inst. 1975, 55, 1253. 7. Ribi, E.; Cantrell, J.L.; Schwartzman, S.M.; and Parker, R. "Augmenting Agents in Cancer Therapy"; Hersh, E.M.; Chirigos, M.A.; Mastrangelo, M.J., Eds.; Raven Press: New York, 1981, pp. 15-31. 8. Ribi, E.; Amano, K.; Cantrell, J.; Schwartzman, S.; Parker, R.; Takayama, K. Cancer Immunol. Immunother. 1982, 12, 91. 9. Takayama, K.; Ribi, E.; Cantrell, J.L. Cancer Res. 1981, 41, 2654. 10. Ribi, E.; Parker, R.; Strain, S.M.; Mizuno, Y.; Nowotny, A.; Von Eschen, K.B.; Cantrell, J.L.; McLaughlin, C.A.; Hwang, K.M.; Goren, M.B. Cancer Immunol. Immunother. 1979, 7, 43. 11. Adam, A.; Cirobaru, R.; Ellouz, F.; Petit, J.-F.; and Lederer, E. Biochem. Biophys. Res. Commun. 1974, 56, 561. 12. Milner, K.C.; Rudbach, J.A.; Ribi, E. "Microbial Toxins. A Comprehensive Treatise"; Weinbaum, G.; Kadis, S.; Ajl, S.J., Eds.; Academic Press: New York, 1971, pp. 1-65. 13. Bottoms, G.D.; Templeton, C.B.; Fessler, J.S.; Johnson, M.A.; Roesel, O.F.; Ewert, K.M.; Adams, S.B. Am. J. Vet. Res. 1982, 43, 999.



10.

T A K A Y A M A ET A L .

Use of Endotoxin

in Cancer

Immunotherapy

233

14. Hinshaw, L.B.; Spink, W.W.; Vick, J.A.; Mallet, L.; Sinstad, J. Am. J. Physiol. 1961, 201, 144. 15. Ribi, E.; Milner, K.C.; and Perrine, T.D. J. Immunol. 1959, 82, 75. 16. Nowotny, A. Nature 1963, 197, 721. 17. Johnson, A.G.; Nowotny, A. J. Bacteriol. 1964, 87, 809. 18. Schenck, J.R.; Hargie, M.P.; Brown, M.S.; Ebert, D.S.; Yoo, A.L.; and McIntire, F.C. J. Immunol. 1969, 102, 1411. 19. McIntire, F.C.; Hargie, M.P.; Schenck, J.R.; Finley, R.A.; Sievert, H.W.; Rietschel, E.T.; and Rosenstreich, D.L. J. Immunol. 1976, 117, 674. 20. Rosner, M.R.; Tang, J.Y.; Barzilay, I.; and Khorana, H.G. J. Biol. Chem. 1979, 254, 5906. 21. Amano, K.; Ribi, E.; Cantrell, J.L. Biochem. Biophys. Res. Commun. 1982, 106, 677. 22. Westphal, O.; Luderitz, O. Angew. Chem. 1954, 66, 407. 23. Qureshi, N.; Takayama, K.; and Ribi, E. J. Biol. Chem. 1982, 257, 11808. 24. Galanos, C.; Lüderitz, O.; Westphal, O. Eur. J. Biochem. 1969, 9, 245. RECEIVED May 31, 1983


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