Generator-Produced Bi-212 - ACS Symposium Series (ACS

15 Generator-Produced Bi-212 Chelated to Chemically Modified Monoclonal Antibody for

Use

in Radiotherapy

Downloaded by GEORGETOWN UNIV on January 7, 2018 | http://pubs.acs.org Publication Date: January 30, 1984 | doi: 10.1021/bk-1984-0241.ch015

O T T O A . G A N S O W — I n o r g a n i c and R a d i o i m m u n e Chemistry Section, R a d i a t i o n Oncology Branch, D i v i s i o n of Cancer Treatment, National Cancer Institute and Laboratory of Chemical Physics, N a t i o n a l Institute of Arthritis, Diabetes, Digestive, and K i d n e y Diseases, N a t i o n a l Institutes of Health, Bethesda, MD 20205 ROBERT W. ATCHER and DANIEL C. LINK—Inorganic and R a d i o i m m u n e Chemistry Section, R a d i a t i o n Oncology Branch, D i v i s i o n of Cancer Treatment, N a t i o n a l Cancer Institute, N a t i o n a l Institutes of Health, Bethesda, MD 20205 ARNOLD M. FRIEDMAN and ROBERT N a t i o n a l Laboratory, Argonne, IL 60439

H.

S E E V E R S — C h e m i s t r y D i v i s i o n , Argonne

WENDIE ANDERSON, DAVID A. SCHEINBERG, and METTE STRAND—Department of Pharmacology and Experimental Therapeutics, Johns H o p k i n s University S c h o o l of Medicine, Baltimore, MD 21205

I t has been demonstrated that monoclonal antibodies may be chemically modified by r e a c t i o n with metal chelates without l o s s of antibody a c t i v i t y or specificity. A radion u c l i d e generator has been made to provide a source f o r Bi-212 to be used f o r attachment of bismuth chelates to antibody. Such antibody-metal-chelate conjugates appear to be s t a b l e i n v i v o and may provide a new method f o r radiotherapy. The development (1) of monoclonal antibodies with very high s p e c i f i c i t y f o r malignant c e l l s suggests that these agents might might serve as t a r g e t i n g v e h i c l e s f o r transport of tracers or c y t o t o x i c agents to tumors f o r use i n diagnosis or therapy (2). Recently Scheinberg, Strand, and Gansow demonstrated that r a d i o a c t i v e metal chelates could be c o v a l e n t l y l i n k e d to monoclon a l antibody without compromising the a c t i v i t y or s p e c i f i c i t y of the p r o t e i n ( 3 ^ ) · Rapid, tumor s p e c i f i c uptake was demonstrated by high r e s o l u t i o n gamma camera images of mouse e r y t h r o i d tumors obtained by using leukemia c e l l s p e c i f i c monoclonal antibodies l a b e l e d with i n d i u m - I l l c h e l a t e s . Radioiodinated a n t i b o d i e s have a l s o been employed f o r i n v i v o studies of p r o t e i n l o c a l i z a t i o n , d i a g n o s t i c imaging, and tumor therapy 02,5). However, the c o v a l ent attachment of iodine to t y r o s i n e residues present i n the antibody f r e q u e n t l y compromises the s p e c i f i c i t y and a c t i v i t y of the immunoprotein. Moreover, degradation of carbon-iodine bonds to f r e e iodine i n vivo i s w e l l known ( 6 ) . Therapeutic p r o t o c o l s would not be optimal because iodine-131 can d e l i v e r only low 0097-6156/84/0241-0215S06.00/0 © 1984 American Chemical Society

Knapp and Butler; Radionuclide Generators ACS Symposium Series; American Chemical Society: Washington, DC, 1984.


216

RADIONUCLIDE

GENERATORS

l i n e a r energy t r a n s f e r (LET) β r a d i a t i o n as w e l l as u n d e s i r a b l e gamma e m i s s i o n s . M o n o c l o n a l a n t i b o d i e s l o c a l i z e o n t h e c e l l u l a r l e v e l a n d a r e i d e a l c a r r i e r s f o r h i g h LET r a d i a t i o n α e m i t t e r s . A s t a t i n e - 2 1 1 h a s a s e v e n h o u r h a l f - l i f e , e m i t s α p a r t i c l e s and has been p r o p o s e d as a u s e f u l r a d i o t h e r a p e u t i c agent (7-9)· A s t a t i n e i s a h a l o g e n and w i l l d i r e c t l y react with tyrosine r e s i d u e s and w i l l be s u b j e c t t o t h e same i n v i v o l i m i t a t i o n s a s iodine including thyroid uptake. In addition, any a s t a t i n e i s o t o p e must be p r o d u c e d by α p a r t i c l e bombardment o f b i s m u t h i n a cyclotron w h i c h imposes s e v e r e c o n s t r a i n t s on t h e g e n e r a l a v a i l a b i l i t y of t h i s radionuclide. L i n k i n g m e t a l c h e l a t e s t o a n t i b o d i e s p r o v i d e s a more v e r s a t i l e method f o r l a b e l i n g a n t i b o d i e s w i t h r a d i o n u c l i d e s . There a r e numerous m e t a l l i c r a d i o n u c l i d e s a v a i l a b l e f o r u s e i n d i a g n o s i s and t h e r a p y w h i c h form complexes s t a b l e i n v i v o . F o r exam p l e , I n - I l l i s a pure γ e m i t t e r i d e a l f o r c o n v e n t i o n a l imaging. I s o t o p e s o f scandium, gallium, copper, palladium or rhenium w o u l d p r o v i d e s o u r c e s f o r l o w LET b e t a r a d i a t i o n . The p r i m a r y f o c u s o f o u r c u r r e n t tumor t h e r a p y r e s e a r c h makes u s e o f B i - 2 1 2 a one h o u r h a l f - l i f e , α e m i t t i n g r a d i o n u c l i d e c o n v e n i e n t l y o b t a i n e d from an e a s i l y used g e n e r a t o r , thus a v o i d i n g t h e problems of c y c l o t r o n p r o d u c t i o n . A R a d i o n u c l i d i c G e n e r a t o r f o r Lead-212 a n d B i s m u t h - 2 1 2 S e v e r a l y e a r s ago Z u c c h i n i a n d F r i e d m a n d e v e l o p e d a l o n g l i v e d g e n e r a t o r f o r Pb-212 and B i - 2 1 2 w h i c h was b a s e d on t h e decay o f Th-228 ( 1 0 ) . I n F i g u r e 1 we show t h e d e c a y scheme f o r Th-228 and daughters. The separation o f the Pb-212 and B i - 2 1 2 r a d i o n u c l i d e s f r o m Th-228 i s b a s e d upon t h e e l u t i o n o f Rn-220 i n the generator. Figure 2 i s a schematic i l l u s t r a t i o n o f the o r i g i n a l generator. The Rn-220 i s e l u t e d f r o m t h e Th-228 and Ra-224 by p u r e w a t e r and d e c a y s t o P b - 2 1 2 w h i c h i s bound b y t h e c a t i o n exchange c o l u m n , and t h e P b - 2 1 2 and B i - 2 1 2 equilibrium m i x t u r e i s e l u t e d from t h e c a t i o n e x c h a n g e column with 2 Ν HCl. A l t e r n a t i v e l y , t h e c a t i o n exchange column i s f o l l o w e d by a bed o f a n i o n exchange r e s i n w h i c h a b s o r b s Pb and B i i n 2 Ν H C l . The Pb-212 i s then e l u t e d w i t h water ( F i g u r e 3 ) . The o v e r a l l y i e l d i s c o n s i s t e n t l y between 80 and 90 p e r c e n t . When t h e f i n a l bed o f a n i o n exchange r e s i n i s u s e d t h e r e was no o b s e r v e d b r e a k t h r o u g h o f Th-228 above t h e 10 ppm l i m i t s o f d e t e c t i o n . I t s h o u l d be noted that these g e n e r a t o r s have a unique p o t e n t i a l as t h e r a p e u t i c t o o l s s i n c e 1 m i l l i c u r i e o f Pb-212 and d a u g h t e r s w i l l y i e l d an i n t e g r a t e d d o s e o f 232,000 r a d s o f h i g h LET r a d i a t i o n and 25,000 r a d s o f l o w LET r a d i a t i o n i f c o n f i n e d t o one m l o f tissue. The g e n e r a t o r was i n i t i a l l y u s e d t o p r o v i d e Pb-212 t o make r a d i o a c t i v e l i p o s o m e s f o r immune s u p p r e s s i o n ( 1 1 ) . The o b j e c t i v e o f t h e s e s t u d i e s was t o s u p p r e s s t h e immune r e s p o n s e p r i o r t o organ t r a n s p l a n t . The f i r s t s t u d i e s w e r e p e r f o r m e d w i t h l e a d - 2 1 2 i n n o n a n t i g e n i c l i p o s o m e s i n r a t s i n o r d e r t o measure n o n s p e c i f i c


GANSOW ET AL.

Generator-Produced

Bi-212


15.

Figure 2. Generator, A = water r e s e r v o i r Β = sodium t i t a n a t e column c o n t a i n i n g Th-228 C = c a t i o n exchange column and Pb-212.


217


218

RADIONUCLIDE GENERATORS

d e s t r u c t i o n of antibody producing Β c e l l s . One day f o l l o w i n g i n j e c t i o n of the liposomes, the animals were challenged with a s e r i e s of antigens and 5 days l a t e r they were assayed f o r antibody production, and the number of s u r v i v i n g Β c e l l s i n the spleen was determined. Figures 4 (11) demonstrates that the non s p e c i f i c r e d u c t i o n of immune response c o r r e l a t e s w e l l with the d e s t r u c t i o n of a l l Β lymphocytes. More recent experiments have i n d i c a t e d s i m i l a r r e s u l t s obtained with phagocytic c e l l s l a b e l l e d with Pb-212 (12). In both cases i t was found that about 10 decays of Pb-212 and daughters would destroy a Β c e l l . The generators used f o r these experiments employ Dowex® 1 and Dowex® 50 i o n exchange r e s i n s i n the e l u t i o n columns. Tho rium-containing t i t a n a t e columns have been used f o r 3 yrs and proven r e l i a b l e , but there has been a general breakdown of the t i t a n a t e i o n exchange m a t e r i a l , probably due to r a d i a t i o n damage, which causes c l o g g i n g and a decrease i n flow r a t e . In order to compensate f o r these problems we have increased the e l u t i o n pressure to about 25 l b s . At that p o i n t , u s u a l l y a f t e r 6-9 months, we have disassembled the generators, r e p u r i f i e d the Th-228, and r e c o n s t i t u t e d new generators. One a d d i t i o n a l compli c a t i o n has been the need f o r a safe f a c i l i t y , such as a glove box, f o r handling the Th-228 generator. Though a glove box has been i n s t a l l e d at s e v e r a l i n s t a l l a t i o n s , i t i s inconvenient and presents a p o t e n t i a l hazard. In order to ameliorate these c o n d i t i o n s we have r e c e n t l y designed and t e s t e d the new, d i s p o s b l e generator, based on the Ra-224 daughter (13) o u t l i n e d schemati c a l l y i n Figure 5. We have a l s o incorporated a set of valves to allow repeated c o l l e c t i o n of the Pb-212 and Bi-212 without un s e a l i n g the generator. This new design has e l i m i n a t e d the need f o r a glove box. In a d d i t i o n , the Ra-224 generators have a u s e f u l l i f e of a few weeks and do not r e q u i r e r e f a b r i c a t i o n . We are now developing an automated system to allow p u r i f i c a t i o n of large amounts of Ra-224 at p e r i o d i c i n t e r v a l s which w i l l allow the c o n s t r u c t i o n of a number of generators f o r c o l l a b o r a t i v e programs. The Chemical M o d i f i c a t i o n of Antibody with Metal Chelates The chemical m o d i f i c a t i o n of antibody by metal chelates may be viewed as r e q u i r i n g two d i s t i n c t but permutable chemical proced ures which i n c l u d e the attachment of a c o o r d i n a t i n g l i g a n d to antibody and the formation of the k i n e t i c a l l y i n e r t metal com plex. Three c l a s s e s of p o s s i b l y u s e f u l ligands are shown i n Figure 6. The s u b s t i t u t e d cryptâtes r e c e n t l y described by Gansow and co-workers are the most complicated to prepare chemically, but could p o t e n t i a l l y carry the most l e t h a l i s o t o p e s , the f i s s i o n able a c t i n i d e s (14,15). Bifunctional ethylenediaminetetraacetic a c i d (EDTA) and d i e t h y l e n e t r i a m i n e p e n t a a c e t i c a c i d (DTPA) d e r i v a t i v e s (16-19) are more e a s i l y synthesized and allow t a r g e t i n g of more t r a c t a b l e isotopes of the group IIIA, VA or t r a n s i t i o n metals as higher valence ions (3^,4).


15.

Générât or-Produced

GANSOW ET AL.

ΙΟΟι 1 1 1 1 1 1 1 — ι i 1—. Downloaded by GEORGETOWN UNIV on January 7, 2018 | http://pubs.acs.org Publication Date: January 30, 1984 | doi: 10.1021/bk-1984-0241.ch015

219

Bi-212

1 1 1 r-

5

1

0.0

J ι I ι I ι I ι L _l 0.2 0.4 0.6 0.8 1.0

I

1.2

I

I

1.4

L.

1.6

ELUTION RATE (ml/min)

F i g u r e 3.

Y i e l d o f Pb-212 as f u n c t i o n o f w a t e r

flow.


RADIONUCLIDE GENERATORS


1009-

5

fth

1.5 Dose of

2 l 2

Pb(/iCi)

F i g u r e 4. R e d u c t i o n o f immune r e s p o n s e b y Pb-212 liposomes.


Generator-Produced

GANSOW ET AL.

Bi-212

Pump I.

H0 Reservoir 2

0.2-2.0 ml/min


Ra - 224 Column 3 mm χ 3mm Biorad D50

Pump 2.

2M HCl Reservoir

2 ml Reservoir for Rn decay

0.5 ml per min

->—Φ 3 way valve Pb-212 Column 3mm χ 5 mm Biorad-D50 WASTE POT

^ 5 3 way valve Sample Vial

Figure 5.

Schematic diagram f o r the Ra-224 generator.

^CHjCOO/ C

R

H COO2

_/ —CH COO"

Ύ

N

M+3

\

C

H

2

2

2

C

O

° "

CH COO"

r ^ C H ^ O O R—Ν Γ

M

+ 3

/CH.COO2

2

^CH COO2

CRYPTANDS

EDTA

DTPA

Figure 6. Three b i f u n c t i o n a l metal c h e l a t e s : sub s t i t u t e d cryptand, b i f u n c t i o n a l EDTA, and DTPA.



222

RADIONUCLIDE

GENERATORS

Metallic radionuclides with s u i t a b l e c h a r a c t e r i s t i c s f o r d i a g n o s t i c nuclear medicine that can be chelated t i g h t l y include the p o s i t r o n emitting gallium-68, and gamma emitting radionu c l i d e s such as technecium-99m (20) and i n d i u m - I l l - These r a d i o n u c l i d e s a l l have short h a l f - l i v e s that vary from about one hour to three days. Isotopes which might have therapeutic e f f e c t s include s e v e r a l beta emitting r a d i o n u c l i d e s of scandium or rhe nium and the alpha emitting r a d i o n u c l i d e s bismuth-212 or a c t i n ium-225. The higher valence ions of each of these elements bind to EDTA of DTPA with pK values >24 (21). Our studies have therefore focused on conjugation of these ligands to antibody. Chemical m o d i f i c a t i o n of p r o t e i n s must occur at a r e a c t i v e f u n c t i o n a l group on the molecular backbone. Three u s e f u l chemi c a l substituents include the phenolic side chain of t y r o s i n e , the ε amino group of l y s i n e and the cysteine s u l f h y d r y l groups. We eliminated s u l f h y d r y l attachment as a candidate because of the w e l l known i n s t a b i l i t y of d i s u l f i d e linkages i n aqueous media, although i f a t h i o e t h e r linkage could be formed, i t might be u s e f u l . Attempts to l i n k l i g a n d to t y r o s i n e by r e a c t i o n of l-(p-benzyldiazonium)EDTA (3,4) with antibody produced only con jugates which had immunoreactivity and s p e c i f i c i t y severely com promised. S i m i l a r l y , conjugates of the p-hydroxybenzimidate of l-Cp-benzyldiazonium)EDTA (18) met the same f a t e , presumably be cause of the high s o l u t i o n pH and s a l t concentration required f o r for e f f i c i e n t r e a c t i o n . The f i r s t s u c c e s s f u l conjugation of chelate to antibody was reported by Scheinberg, Strand and Gansow i n 1981 (3^»4)· m o d i f i c a t i o n of the method of Yeh, et a l (16-19) was employed to prepare l-(p-carboxymethoxybenzyl)EDTA and i t s i r o n ( I I I ) com plex formed by a i r o x i d a t i o n of ferrous s u l f a t e i n aqueous s o l u t i o n s of the l i g a n d . Coupling through the carboxylate to form and a c i d amide linkage with ε amino groups of antibody l y s i n e s was e f f e c t e d by the water s o l u b l e 1,3-(3-dimethylaminopropyl)carbodiimide. By use of iron-59 t r a c e r , i t was determined that approximately 0.9 chelates were bound to immunoprotein. Iron was subsequently removed by d i a l y s i s against a buffered s o l u t i o n of ascorbate, l e a v i n g the EDTA p o r t i o n of the molecule free to be reacted with another metal i o n . Antibody-chelate-metal conju gates were formed with indium, g a l l i u m , and scandium by r e a c t i o n of the appropriate t r i v a l e n t i o n . No l o s s of antibody a c t i v i t y or s p e c i f i c i t y was measured by c e l l binding study. In more recent experiments, two d e r i v a t i v e s of DTPA have proven to be the simplest to prepare and most widely u s e f u l conjugating lig ands. Both the carboxycarbonic anhydride of DTPA (17) and the DTPA dianhydride (16,19,22-25) react e a s i l y with antibody near n e u t r a l s o l u t i o n pH values to give conjugates with no l o s s of immunoreactivity or c e l l binding s p e c i f i c i t y and are u s e f u l f o r imaging, Figure 7. We therefore have studied t h e i r r e a c t i o n with an e a s i l y obtained immunoprotein, bovine IgG. The r e s u l t s of pH and concentration dependence of l i g a n d - p r o t e i n conjugation are shown i n Figures 8-10. In general, e f f i c i e n c y of conjugation i s A



15.

GANSOW ET AL.

Generator-Produced

223

Bi-212

F i g u r e 7. Images o f n o r m a l a n d l e u k e m i c m i c e i n j e c t e d w i t h l n - 1 1 1 D T P A - l a b e l e d m o n o c l o n a l a n t i b o d i e s . A , Β ( n o r m a l ) a n d C,D ( l e u k e m i c ) were i n j e c t e d w i t h c o n t r o l (A,C) o r t u m o r - s p e c i f i c (B,D) a n t i b o d y . R e p r o d u c e d w i t h p e r m i s s i o n f r o m R e f . 4. C o p y r i g h t 1982, A m e r i c a n A s s o c i a t i o n f o r t h e Advancement o f S c i e n c e . 40

1200 [Chelate]/[Protein] in Reaction of IgG with DTPA Dianhydride F i g u r e 8. C o n c e n t r a t i o n dependence of r e a c t i o n of DTPA dianhydride with bovine IgG.


RADIONUCLIDE

GENERATORS


2.0

Ο,ο

I

•

0

«

100

1

200

«

300

1

400

J

500

600

[Chelate]/[Protein] In Reaction of IgG with DTPA t-Butylcarboxycarbonic

Anhydride

Figure 9. Concentration dependence of r e a c t i o n of DTPA i-butylcarboxycarbonic anhydride with bovine IgG.

1.4

O.o

ι

1

0

1

1

1

' 2

4

1

» 6

1

' 8

r

1

1

1

» 10

12

14

pH of Reaction of IgG frith DTPA Dianhydride at [Chelate]/[Protein] = 100:1

Figure 10. pH dependence of r e a c t i o n of bovine IgG with DTPA dianhydride.



15.

GANSOW ET AL.

Generator-Produced

Bi-212

225

maximized at a s o l u t i o n pH near 8.0. This can be explained by an a n a l y s i s of the chemistry of the conjugation. Reaction of the anhydride with ε amino groups of l y s i n e w i l l be favored by deprotonation i n basic s o l u t i o n . Aqueous decomposition of the anhydrides i s a c i d or base catalyzed, so i t i s not s u p r i s i n g that the conjugation r e a c t i o n proceeds best under m i l d l y basic c o n d i t i o n s . Increasing anhydride concentration increases the number of ligands attached to immunoprotein. In a d d i t i o n , high numbers of attached ligands o f t e n adversely a f f e c t the s p e c i f i c i t y or r e a c t i v i t y of conjugated monoclonal a n t i b o d i e s . The l i n k i n g of a metal to an antibody could, i n p r i n c i p l e , be accomplished by forming the metal chelate e i t h e r p r i o r to or a f t e r attachment to p r o t e i n . Success to date has been achieved only by formation of the p r o t e i n - l i g a n d conjugate before metal c h e l a t i o n . The complexation r e a c t i o n has s e v e r a l general f e a t u r e s . F i r s t , r e a c t i o n s between the m e t a l l i c r a d i o n u c l i d e s and antibodies are almost always performed with sub-stoichiometric q u a n t i t i e s of chelate and metal i o n . I t i s therefore of the utmost importance that no c a r r i e r added metals obtained from commercial sources be exceedingly pure or e l s e be purified p r i o r to use. Reactions of " c a r r i e r added" metal s o l u t i o n s are not l i k e l y to be of use because of the ease with which a v a i l a b l e chelate s i t e s become saturated. Because the formation of chelate complexes i s u s u a l l y a bimolecular r e a c t i o n , the complexation w i l l proceed o p t i m a l l y when more c h e l a t i o n s i t e s are a v a i l a b l e . S i m i l a r l y , the more isotope i n s o l u t i o n , the f a s t e r the r e a c t i o n . Employment of a c a r r i e r chelate to insure s o l u b i l i z a t i o n of the radiometal i s of value to maximize a v a i l a b l e isotope and the acetate i o n has proven u s e f u l . Imaging and Therapy Perhaps the greatest p o t e n t i a l d i a g n o s t i c use for r a d i o l a b e l e d monoclonal antibodies i s tumor imaging. A number of r a d i o h a l i d e s and radiometals have been suggested f o r t h i s purpose (23). A n t i body conjugated DTPA complexes have proven to be the most u s e f u l because of the v a r i e t y of metals usable, the r e l a t i v e ease of p r o t e i n conjugation, the r e t e n t i o n of b i o l o g i c a l a c t i v i t y , and the s t a b i l i t y of the conjugate i n v i v o . We have demonstrated that high r e s o l u t i o n images of tumors can be obtained using radiometal chelate conjugated tumor s p e c i f i c antibody (3>4)· S i m i l a r studies with 1-131 labeled antibody did not produce usable scans (24). In recent gamma camera s t u d i e s performed at the N a t i o n a l Cancer I n s t i t u t e , i n d i u m - I l l l a b e l l e d s p e c i f i c a n t i body was a l s o seen to provide images of s o l i d human breast tumor t i s s u e s grown i n athymic (nude) mice (25). Extension of these studies to human diagnosis i s now underway. Scheinberg and Strand (26) have addressed the therapeutic e f f e c t s of a t u m o r - s p e c i f i c antibody against the Rauscher e r y t h r o leukemia. A dose response c o r r r e l a t i o n between t h i s antibody and tumor i n h i b i t i o n was measured. The dose response curve


r a d i o n u c l i d e


226

generators

served then as a baseline f o r q u a n t i t a t i o n of the therapeutic e f f e c t s of c y t o t o x i c r a d i o n u c l i d e s conjugated to the antibody. Scheinberg and Strand reported that i n t h i s model system 1-131 l a b e l e d antibodies are not more e f f e c t i v e than the unlabeled antibody i t s e l f . This lack of therapeutic e f f i c a c y may result from the long range of the beta p a r t i c l e s emitted, the long h a l f - l i f e of the nuclide as compared to the r a p i d catabolism of the c e l l bound antibody, or to the low density of r a d i a t i o n received by the tumor c e l l s (20). P o t e n t i a l l y more c y t o t o x i c are the α emitting r a d i o n u c l i d e s such as bismuth-212 which i s obtained from a generator. This radiometal may be conjugated by antibodies d e r i v a t i z e d with EDTA or DTPA l i g a n d s . The Bi-212 nuclide has a one hour h a l f - l i f e and emits α p a r t i c l e s with a range of 40-90 microns. I f Bi-212 had been s u b s t i t u t e d f o r I n - I l l i n the leukemic tumor imaging experiments described above, s e v e r a l thousand rads of high LET r a d i a t i o n would have been d e l i v e r e d to the tumor, enough to insure t o t a l therapy. The s t a b i l i t y of bismuth DTPA chelates has been i n v e s t i g a t e d in vivo. Both f r e e bismuth and the complex were i n j e c t e d i n t o normal and leukemic mice. As a n t i c i p a t e d , the f r e e metal was r e t a i n e d i n s u b s t a n t i a l quantity i n the l i v e r and other organs, whereas the chelated metal c l e a r e d r a p i d l y through the kidneys. C e l l k i l l i n g by Bi-212 l a b e l e d antibodies has been i n v e s t i g a t e d in vitro. Therapeutic potency was high despite the low s p e c i f i c a c t i v i t y c u r r e n t l y a v a i l a b l e (27). Experiments addressing the s p e c i f i c i t y , s t a b i l i t y and t h e r a p e u t i c potency of Bi-212 antibody conjugates i n v i v o are i n progress. Ackn owle dgment s Research support from the N a t i o n a l Cancer I n s t i t u t e , Grant NCIN0ICP81052 i s acknowledged. Work at Argonne N a t i o n a l Laboratory was performed under the auspices of the O f f i c e of Basic Energy Sciences, D i v i s i o n of Chemical Sciences, U.S. Department of Energy under contract W-31-109-ENG-38. Literature Cited 1. 2. 3.

4. 5. 6.

G. Kohler and C. M i l s t e i n , Nature (Lond.), 1975, 256, 495. D.M. Goldenberg, Ed., Cancer Res., 40 (No.8), 1980, Part 2. D.A. Scheinberg, M. Strand, and O.A. Gansow, i n Mono c l o n a l Antibodies i n Drug Development, T.S. August, Ed., American Society for Pharmacology and Experimental Thera p e u t i c s , Bethesda, MD., 1981. Science, 1982, 275, 511. S.E. Order, J.L. K l e i n , D.E. Hinger, P. Alderson, S. S i e gelman, and P. Leichner, Cancer Res., 1982, 42, 44. W.F. Bale, M.A. Contreras, and E.D. Grady, i b i d . , 1980, 40, 2965.


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A.M. Friedman, M.R. Zalutsky, W. Wung and F. Buckingham, P.V. Harper, J r . , G.H. Scherr, R.W. F i t c h , F.P. Stuart, and S.J. Simonian, I n t . J. Nucl. Med. and B i o l . , 1977, 4, 219. J . Smit, J . Myburgh and R.Neirinckx, C l i n . Exp. Immunol., 1973, 14, 107. W.D. Bloomer, W.H. McLaughlin, R.D. N e i r i n c k x , S.J. A d e l s t e i n , P.R. Gordon, T . J . Ruth and A.P. Wolf, Science, 1981, 212, 340. G.L. Z u c c h i n i and A.M. Friedman, I n t . J . Nucl. Med. B i o l . , 1982, 9, 83. M.K. Rosenow, G.L. Z u c c h i n i , D.M. B r i d e w e l l , F.D. S t u a r t , and A.M. Friedman, I n t . J. Nucl. Med. B i o l . , i n press. F.P. S t u a r t , p r i v a t e communication. A.M. Friedman, R. Atcher, and R. Seevers, to be p u b l i s h e d . O.A. Gansow, R Kausar, and K.B. T r i p l e t t , J. H e t e r o c y c l i c . Chem., 1981, 18, 297. T. Peterson, Ph.D. t h e s i s , Michigan State U n i v e r s i t y , East Lansing, MI, 1982. S.M. Yeh, D.G. Sherman and C.F. Meares, A n a l . Biochem., 1979, 100, 152. G.E. Krejcarek and K.L. Tucker, Biochem. Biophys. Res. Commun., 1977, 77, 581. C. Paik, D.E. Herman, W.D. Eckelman and R.C. Reba, J. Radioanal. Chem., 1980, 57, 533. J . Dazz, Switzerland Patent 17930, 1966. B r i t i s h Patent 1,161,461, 1967. U.S. Patent 3,660,388, 1972. B.A. Khaw, J.T. F a l l o n , H.W. Strauss and E. Haber, Science, 1980, 209, 295. S t a b i l i t y Constants of Metal-Ion Completes, Chemical Soci e t y Sp. Publ. Nos. 17, 25, Chemical S o c i e t y , London, 1964, 1971. IUPAC Chemical Data S e r i e s , No. 22, Pergamon Press, New York, 1979. D.J. Hnatowich, W.W. Layne and R.L. C h i l d s , I n t . J . Appl. Radiat. Isotopes., 1982, 33, 327. D.A. Scheinberg, M. Strand and O.A. Gansow i n C e l l Fusion, E.G. Basset, Ed., Raven Press, 1983. D.A. Scheinberg, W. Anderson, and M. Strand i n Radioimmuno -imaging, S. B u r c h i e l and B. Rhodes, Eds., E l s e v i e r , N.Y., 1982. O.A. Gansow, D. Colcher, and J . Schlom, to be published. D.A. Scheinberg and M. Strand, Cancer Res., 1982, 43 265. M. Strand, D.A. Scheinberg, W. Anderson, O.A. Gansow and A.M. Friedman, Proceedings of the Armand Hammer Symposium, 1983, i n press.

8. 9.

10.


11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

22. 23. 24.

25. 26. 27.

RECEIVED

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O c t o b e r 27, 1983


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Generator-Produced Bi-212 - ACS Symposium Series (ACS

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