RICIN-ANTIBODY CONJUGATES

Abstract: A conjugate of ricin being whole ricin or a derivative of ricin, which derivative per se being inhibitory of protein synthesis in cell free systems, with an antibody which is preferentially absorbed by a tumour cell as compared to a non-tumour cell and wherein the galactose binding site of said ricin is blocked and pharmacologically acceptable salts thereof.

Description: RICIN-ANTIBODY CONJUGATES This invention relates to a chemical compound. In a particular aspect this invention relates to a ricin-antibody conjugate. The plant toxin ricin has been invol ved in extensive research since the 1950's when it was suggested that it may be a useful anti-tumor agent because of its great potency. Ricin has a molecular weight of approximatel 67,000 and is composed of two polypeptide chains, referred to as the A and 8 chains, which are disulphide lin ed. Ricin has a strong binding affinity for simple sugars and an even stronger affinity for o 1 i g o s a c c h a r i d e s bearing D-galactose or N - acetyl galatosamine as terminal residues. It is through such molecules on the cel l surface that ricin attaches to the membranes of the cells. Thus in the absence of an inhibitory mechanism whole ricin is toxic to al l cel ls. Such binding and toxicity can therefore be co e i i ely inhibited in vitro by the presence of lactose or D-galatose. The problem of specificity of ricin in anti-tumor therapy is currently being tackled by covalently binding it to immunog 1 obu 1 ins to form hybrid molecules known as immunotoxins. In this way it is hoped that the ricin may be directed only to those cells specifical ly reacting with the i mmu n o g 1 o b u 1 i n of the im unotoxin. When the whole toxin has been conjugated to antibodies there has been a strong residual non-specific cytotoxicity due to the binding capacity of the 8 chain of the toxin. As a consequence research has been concentrated on coupling the A-chaiπ of the whole ricin molecules, which is responsible for inhibition of protein synthesis or ribosomes. U n f o r t u n t e 1 y , A-chain immunotoxins have been disappointing and it is quite clear that the B chain is necessary for ricin to be a potent poison. The present invention provides a conjugate of ricin being whole ricin or a derivative of ricin, which derivative per se being inhibitory of protein synthesis in cel l free systems, with an antibody which is preferentially absorbe by a tumour cel l as compared to a non tumour cel l and wherein the galactose binding site of said ricin is blocked and pharmacologically acceptable salts thereof. Preferably the conjugation is via, a disulphide linkage. The present invention a lso provides . a compound of formula Ab-Y-X 1 -NHR 1 where Ab and the Y group attached thereto represent an antibody radical and wherein Y is S or NH, R ι and the a ino group attached thereto represent a whole ricin radical , and X^ , or Y-X (when Y is S), represent a conjugating chain including a disulphide linkage. In a pref erred instance , the present inv ention provides compounds of formula X, fll -/_<_-<: - . <-.)„ -5-S-(c )~c- N H- . wherein Ab and the amino or sulphur to which it is attached represents antibody radical, R and the amiπo group to which it is attached represents a whole ricin radical , Z. and Z 2 which may be the same or different are H, alkyl , aryl , carboxyl , halide, cyano, ester, carboxamide, sulphoπate, hydroxy or amino, n .. and π 2 which may be the same or different are 1 -10 X- and X- which may be the same or different are NH , 0 or S, and z is 0 or 1 and salts thereof. In a preferred instance, the present invention provides compounds of formula Ab-S-S-R 2 -NH-R 1 II Ab-NH-R 3 -S-S-R 2 -NHR 1 III wherein Ab-S-and Ab-NH- represents an antibody radical, a whole ricin radical and -R 2 NH an d - N ' H-R j which may be the same or different are of formula «. 2 l _ H ' I t/ -wH- c - ( __)„- • — wherein X ή is 0,5 or NH, n is from 1 to 10 and Z 3 is H, alkyl, aryl, carboxyl, halide, cyamα, ester, carboxamide, sulphonate, hydroxy or amino. Particularly preferred compounds are those of formula V 1 and VI below: 2 O ^V OH P , - /ti- -cH*-c - -≤-S " ~Sr" Λ W/3i 5 tti_rC 2 -S- ζ-CH_- c H z - I - NH Ak J_ 8 9 0 with galactose blocking as aforesaid. 1 The present in v ention a l so prov ides the intermediate 2 compounds 3 4 5 6 7 8 9 0 1 2 wherein R^ , Ab, X , X- j , Z_. and Z , n_ j and n have the values 3 given above. 4 Compounds in accordance with this invention can be 5 p re p ar e d b y r ea c t in g wh o l e r ic i n wit h s u cc i n imi dy l 6 p y r i d 1 d i t h i o - p r o p i o n a t e (SPDP) or an ana logue thereto 7 appropriate to the compound desired to obtain a whole ricin 3 conjugate containing a- -S-S- group , reacting an antibody with SPDP or an analogue thereto appropriate to the compound desired to obtain an antibody conjugate containing a -S-5- group, reducing one of the conjugates to form a -SH group and reacting the reduced conjugate with the other of the conjugates to obtain the compound desired. Other methods of obtaining compounds in accordance with this invention include using S - a c e y 1 m e r c a t o s u c c i n i c anhydride ( S A PI S A ) or sulphydryl introducing compounds to produce a side chain on an antibody or whol e ricin containing a -S- linkage, reducing and then reacting to form an antibody whole ricin conjugate containing a -S - S - linkage. Intermediate VII can be prepared when ricin is reacted with SPDP. Intermediate IX can be prepared (X 2 = NH Z * - H n_ j _- 3) by reacting Ab with i inothio 1 ane. Intermediate VIII can be made by treating Ab with di hiotheito 1 to reduce the intrachain disulphide bonds. Compounds of formula VI are best prepared using SPDP but with the caution that if the pH is not above 8, preferably pH about 7.5, in the absence of galactose or lactose an unacceptably high proportion of product may not have the galactose binding site blocked. Product which has the galactose binding site blocked may be separated from that in which the galactose binding site is not blocked in any convenient way but in general it is preferred that such separation be effected by reaction with a compound which will substantially selectively react with and immobilize product in which the galactose binding site is not blocked. A preferred method of effecting this is to use a column as explained later. Antibodies useful in the present invention included those showing specificity for breast, brain, melanoma, lung, pancreas and colon tumours. The compounds of this invention may be combined with pharmaceutically acceptable carriers. The mode of administration of the compounds of this invention will be as selected. In particular, the compounds of this invention may be administered intravenously, intraperitonealy , intrapluraly, intrapericardia l y, iπtracerebo spinal fluid and directly intratumour. Drawings and Tables forming part of this s ecifica ion are 5 - Formula Drawings - 1 Sheet Part A : - Figures: A1a, A1b, A2, A3a and A3b Part B:- Figures: B1 , B2a, B2b, B3, B4, B5, B6, B7a, B7b, B8, B9a, B9b, B10a, B10b, B10c and B11 Table: B1 , B2, B3 and B4 Part C : - Figures: C1 a, C1 b, C3, C4, C6a, C6b, C6c, C7, CBa, C8b, and C9 Table: C1 and C2 Part D:- Figures: 01 a, D1 b, D2a, D2b, 02c, D5, D7, 08 and 09 Table: 01 £Sll __ Conjugates in accordance with this invention appear not to bind "non-specifically" to target cells and do not require presence of lactose to inhibit any non specific binding (Fig. A1 a and A 1 b ) . Ul h i 1 e these studies- are of interest for in vit££ studies in that lactose does not have to be used (of no great consequence) the major advantage of the non-specific binding sites is that whole ricin antibody compl exes can now be used in ^vo where such high concentrations of lactose cannot be achieved. Thus, mice bearing tumours were treated with three different regimes of whole ricin antibody conjugates. In the first, the ITT(1 ) 75 NS E3 thymoma was growing in the peritoneum of mice in the ascites form and whole ricin antibody conjugates were delivered into the ascites fluid (Fig. A2). when 1 x10 5 tumour cells were given and one injection of ricin-antibody (1 day after tumour inocu l ation) the tumours were erradicated and mice survived indefini el . By contrast those receiving a mixture of antibody and ricin alone died rapidly from ricin poisoning, and those receiving no treatment died shortly thereafter from the tumour. Thus, the whole ricin-antibody con ugates were capable of erradicating tumours from mice in vivo. However, it should be noted that the therapeutic range of the ricin-antibod complex was narrower to greater doses (>0.1 mu g) of ricin-antibody complexes led to the death of the mice due to toxicity and lower doses (<0.05mu g) led to death from the tumour. However, the dose range is not very different to that used for most cytotoxic drugs in the treatment of cancer. In t he second mode l , the s ame tumour grown subcutaneous 1y and measuring 0.6cm in diameter, received two injections of antibody-ricin conjugate intravenously (0.1mu g ricin, 0.45mu g CloAb, on days 8 and 11 after tumour inocula ion). A significant inhibition of tumour growth was observed in the group which received the specific ricin- antibody when compared to other controls which included an irrelevant ricin-antibody complex (Fig. A3). In the third model - perhaps the most dramatic - tumours 0.6cm in diameter growing in the subcutaneous tissue of mice received two injections of ricin-antibody conjugate directly into the tumour (approx 0.2mu g ricin, 0.9mu g l-l 0Ab on days 7 and 9 after tumour inoculation). Within 24 hrs there was a notable difference in size of tumours (Fig.A3b) compared to that of controls and by 48 hrs the treated group had virtually disappeared. Thus, in these three different models whole ricin- antibody conjugates led to either erradication of the tumour or a marked dimunition in tumour size. Ricin would appear to have a lot to offer as a cytotoxic agent because of its great degree of potency - it is estimated that only one toxin molecule is apparently sufficient to kil l one cel l. Prior to these studies, use of ricin irj was impossible b ecaus e o f i t s h igh t o x ic i ty. H owe v e r , w ith c a r e f u l attention to dose and coupling procedures, it now appears that ricin may be suitable for consideration as a therapeutic agent for cancer in man. £XA__£_____I Spec ific c ytotoxic a cti v ity o f who l e r icin immunotoxins. 5mg of affinity purified anti-Ly-2.1 (5.7mg/ml was thiolated with a 5 fold excess of SPDP (2mg/ml in dioxane). 3mg of ricin ( 2 m g / m 1 ) had a trace amount of I - ricin added and was thiolated with a 10 fold excess of SPDP. Both were reacted for 30 ins at room temperature and dialysed against 0.011*1 borate buffer containing 0.151-1 NaCl. The thiolated anti-Ly-2.1 was dialysed for 6 hrs against 0.2I-I Na acetate buffer pH 4.6 with 0.151. NaCl and 2 m l-l EDTA when it was reduced with DTT (3. ml. final concentra ion). The reduced PI o A b was dialysed against deoxygenated borate buffer for 6 hrs. The thiolated ricin was then added in a 1 :1 molar ratio and the mixture al lowed to react for 2 hrs when the reaction was stopped with - e th y 1 ma 1 e imid e. The mixture was purified on a sephacry 1 -S-300 column. The anti- L y-2.1 -r icin immunotoxin was collected and passed down a l actose sepharose column where ~ S % of the reco v ered immunotoxins did not bind thereby separating out poduct with unblocked galactose binding site. Iest_I The cytotoxicity of the purified immunotoxin was examined in the presence (Fig. A1 a) or absence (Fig. A1 b) of 100 m l-l lactose. 8x10 5 ITT(1 ) 75NS E3 cel ls were incubated with different concentrations of toxin or immunotoxin for 1 hr. The cel ls were washed three times to remove any unbound material and resuspended with leucine free R P l-l I and then pulsed with 3 H-leucine (luCi) for 16-24 hrs. Results are expressed as the % inhibition of H-leuciπe incorporation in controls (calls treated with PBS). Iest_II Survival experiment: groups of 10 ( B 6 8 AL B /c ) F . mice were injected i.p. with 1x10 5 ITT(1 ) 75NS E3 cells. Mice received one of the following treatments i.p., one day after tumour - Aθ - ( inoculation: P8S; ricin + anti-Ly-2.1 ; an i -Ly-1.1 -r ici n ; or a π t i -L y - 2.1 - r i c in . Each mouse received 0.05 m u g ricin and 0.22mu g anti-Ly-2.1. Survival was followed for 130 days. ' Results are shown in Fig. A2. Iest_III Tumour growth experiment: Groups of 10 ( 86 x BA LB /c ) F . mice were injected s.c. with 4x10 s ITT( 1 ) 75NS E3 cells, mice received the following treatments: PBS; anti-Ly-2.1: anti- Ly-1.1 -ricin; and anti-Ly-2.1 -ricin. Each mouse received 0.1mu g ricin and 0.45 mu g anti-Ly-2.1 intravenousl (Fig. A3a) on days 6 and 8 after tumour innoculation or directly into the tumour(Fig A3b) on days 7 and 9. Tumour growth was calculated as the product of two per endicular diameters. Results are shown in Fig. A3. - 81 - ( .) Part B This part reports on the chemica l l inkage of ricin to M o A b via a disulfide bridge with the use of two bifunctional c r o s s 1 i n k e r s. The coupling procedure l ed to a steric blocking of the galactose binding ability of the B-chain. A n u m b e r o f d i f f e r e n t fl o A b , w i t h t h r e e d i f f e r e n t immunoglobulin isotypes have been conjugated and al l have retained cytotoxic activity but were devoid of galactose binding. MATERIALS AND METHODS lH_!2-_ ££ ■ _.__£ BU5147 (10), EL4 (11 ) and sev eral cl onal variants of the murine thymoma ITT(1 )75NS (12) were grown in . Q h ea t - in a c t i v a t e d newborn ca l f serum in Dulbecco's Modified Eagle medium (DMEM). The human T-cal l leukemia line CE I. ( 13 ) was maint ain ed in P fl I-1640 wit h the s ame additives. ϋ_2____ foAb used were: anti-Ly-2.1 (lgG1 , IgG2a) (14), anti-Ly-1.1 ( I g G 2 a ) (15), which recognize the murine 1 ymp h o c y t e ' a 11 o a n t i g e n s Ly-2.1 and Ly-1.1 res ectively HuLy-m9 (IgG1) (16), an antibody which reacts with the human transferrin receptor; 250-30.6 (IgG2b) (17), an antibody directed against an antigen present on human colon carcinoma and secretory epithelium; 17.1/2 (lgG2a) (18) MoAb directed against an antigen present on human breast tissue; anti-HLA (IgG2a) (19) a monomorphic HLA FloAb. The anti-HLA and 17.1/2 were purified by affinity c h r om a t o g r a p hy or by Protein-A- Sepharose (Pharmacia). ££__.___£________£__£ __£__.__£__££____££ 9. _U£___! Modification of the carbohydrate in ricin was achieved using a mixture of sodium metaperiodate (NalO. ) and sodium cyanoborohydride (NaCNBH 3 ) according to the procedure described by Thorpe et al (21 ). Briefly, the method was as follows, 10 g ricin (1.8 mg/ml, Sigma Chemical Company St. Louis, MO, USA) in 0.2 M sodium acetate buffer pH 3.5 was mixed at 4°C in the dark with an equal volume of a mixture of 20 m NalO^ and 40 mM NaCNBH 3 . After 1 hour incubation 20J (v/v) solution of glycerol (100 mM final concentration) was added to the reaction mixture to destroy unreacted 10. " . The sample was kept on ice for 16 to - 82 - (ι β . 24 hours and was dialized against 0.01 M phosphate buffer containing 0.15 M NaCl pH 7.5. Since HCN is produced during the reaction it was carried out inside a fume cupboard and the dia lysates were disposed of with appropriate precautions. The modified ricin was concentrated with an Amicoπ Concentrator (Amicon corporation, Massachusetts, USA) and stored in the phosphate buffer at -20°C. The significance of carbohydrate modification will be discussed in detail in Part C. __ _ϊi£i2__£i___H3 £_L £__£____ H__ ____£____££"__ Ricin (100 mug, 1 mg/ l) or ricin (I0 4 ~/CNBH 3 ~ modified) was labeled using chlora ine T (22) in the presence of 100 mM lactose and MoAb (100 mug, 1 mg/ml) using the same procedure. Ill£ £_.££ ££_.._£_. £_L !_.__£ ______ __!_---_ __£_--_ ££_____-££-_£ §£____££ ££_------_££__ ! Three buffers were used during the preparation of the conjugates: (a) 0.01 M sodium phosphate, pH 7.5, containing 0.15 M NaC1 ; (b) 0.01 M sodium borate, pH 8.0, containing 0.15 M NaC1 ; (c) 0.2 M sodium acetate, pH 4.6, containing 0.15 M NaCl and 2 M EDTA. __il£__£ ]_,__£___! ££_.__£__£.__££ : f-oAb were coupled to ricin by 2 dif f erent procedures that inv o l v ed the use of heterob i f unct iona 1 protein crosslinkiπg reagents - a novel method described in this part using the h e t erobif unc t iona 1 reagent SAMSA, and a method using SPDP. £££_ii_-- --_--£__. __£___12 -_A______ ! Disulfide linked ricin- antibody conjugates were prepared as follows: (a) Thiolation of MoAb: MoAb (10 mg, 5 mg/ml) in phosphate buffer was treated with a 5-fold excess of SAMSA (Sigma) for 30 minutes and excess reagents were removed by dialysis into phosphate buf f er containing 2 mM EDTA; (b) Thio l ation of ricin/modif ied ricin ( I 0 4 " / B H 3 " modified): 5 mg ricin (1.8 mg/ml) or ricin 10^ " /BH 3 ~ modified) in the phosphate buffer was treated with a 10-fold excess of SPDP (Pierce Chemical Company, Illinois, USA) for 30 minutes and dialysed into phosphate buffer containing 2 mM EDTA for 16 to 24 hours; (c) coupling of ricing to MoAb: The modified MoAb was then treated with 200 mul h y d r o x y 1 a m i n e (1 M in PBS pH 7.0) and after 30 minutes at room temperature it was mixed with the - 8 3 - (ι » . SPDP modified ricin (in a 1 to 1 molar ratio) and allowed to react for 1 to 6 hours. The reaction was terminated by the addition .of N-ethy 1 ma 1 iimide (6.6 mM final concentration), allowed to stand a further 1 hour and then subjectd to gel permeation chromatog r a ph y on Sephacryl S-300 equilibrated with phosphate buffer containing 20 M lactose and eluted with the same buffer. To identify the conjugate fractions, trace amounts of 125 - - 1 _ b e 1 e d ricin (specific activity = 4000 cpm/mug) were added; the profile of the absorbancε measured at 280 nm of the elute is shown in Figure B1 together with the radioacti ity. Fractions 97-105 contained material of Mr 190,000 - 240,000 (calculated from the elution volume), and comprised IgG and ricin in the molar ratio 2:1 , (calculated from measurements of absorbance at 280 nm and radioactivity) and were shown by sodium dodecyl sulfate gel analysis to contain as the major species the 210,000 Mr conjugate (consisting of one molecule of antibody linked to one molecule of ricin) and unconjugated MoAb. Fractions 97-105 and 125-133 (unconjugated ricin Mr65,000) were analysed further by affinity c h r o m a t o g r a p h y (see below). The chemistry of the thiolation and coupling is illustrated in Figure B2a. ££___i.H3£-___£__ U ___l__ §£__£ s Ricin was coupled to MoAb using the heterobifunctiona 1 cross 1 inkinbg agent SPDP. This method couples the proteins by the introduction of thiol groups which can be reacted to form stable disulfide linkages between proteins. SPDP was dissolved in dioxane to give a 2 mg/ml solution. Given amounts of SPDP were reacted with ricin or MoAb in phosphate buffer pH 7.0 or borate buffer pH 8.0 at room temperature for 30 minutes in the quantities shown in Table B1. The number of SPDP residues per molecule was previously determined by calculating the liberation of pyridine-2-thioπe residues from the SPDP molecule under reducing conditions. Release of p y r i d i n e - 2 - t h i o n e was measured by monitoring increasing absorbance at 343 nm on a Pye-Unicam SPB-400 spectrophotometer. Following 30 minutes incubation, thiolated MoAb and ricin were dialysed against the borate buffer for 16 to 20 hours at 4°C. The thiolated - B 4 - (ι MoAb was dialysed against the acetate buffer for 2 hours, and then the SPDP thiol group on the MoAb molecule was reduced with DTT (3.4 mM final concentration) for 30 minutes at room temperature, and further dialysed against deoxygenated acetate buffer for 8 to 10 hours to remove uπreacted DTT. Deoxygenation of the acetate buffer prevents oxidation of the sulfhydryl residues. The immunotoxin was then found by reacting thiolated ricin with the thiolated MoAb for 2 hours at room temperature. The reaction was terminated with the addition of N - e thy 1 ma 1 eimi de (6.6 M final concentra ion). The mixture was allowed to stand at room temperature for 1 hour, and the subjected to gel permeation ch r omat og r ap hy on Sephacryl S-300 as described previously. The chemistry of the SPDP thiolation and coupling is illustrated in Figure B2b. The concentration of ricin in the conjugate fractions was determined from the radioactivity, whereas the concentration of MoAb was ascertained by measuring the absorbance at 280 nm and subtracting the calculated absorbance due to the ricin (using absorption coefficients -_ *ι 1 c-.,m_. = 11.8 for ricin and 14.0 for Ig 3 G)'. __£_. £.__£££ §iπi___.££ ____._____1____X J TlU0 different matrices were employed to further purify the immunotoxins based on the affinity of ricin for Sepharose and were used to determine whether the coupling procedure altered the binding specificity of the bound ricin (conjugated) and free ricin (unconju ated). Conjugate fractions 97 to 105 (see Figure B1) and unconjugated ricin, fractions 125 to 133 (Mr65,000), were pooled and extensively dialysed against the phophate buffer. Lac to s e -S epharos e and Sepharose - 4B were used to determine the galactose binding ability of the immunotoxin and free (unconjugated) ricin. The immunotoxin and ricin fractions were absorbed onto columns at 4°C and also at room temperature. The solutions were passed through the column 3 times, the columns washed with phosphate buffer to remove the material that did not bind and the bound protein was then eluted with phosphate buffer contain 100 mM lactose. ££!!££_-___--_--£ _-i-_-_-_-l£ __!2iX ! The Ly-2.1 MoAb was tested - B 5 - 0 _ ) on the Ly-2.1 + ce l l l ine E2 after each l abel ing for alteration in binding ability. Binding was assessed by comparison of modified (SAMSA treated) and untreated aπti- ly-2.1 in their ability to compete with a n o π s a t u r a t i n g amount of 125 j -anti-Ly-2.1 for binding to 2- 4 x10 5 cells; the CEM cel l line was used as a πonreactive control. In this assay, 25 mul of the untreated or thiolated MoAb (1 mg/ml in 0.5. BSA) was serial ly diluted and iodinated MoAb (2x10 5 cpm/well) was added to each well. The cells were then added, incubated for 30 minutes on ice and πonbound material was removed by washing. The cel l pel lets were counted and the results expressed as the percentage inhibition of the maximum counts bound. £££_.£__, ___.Il----------£ __£I£__2 S The inactivation of cellular protein synthesis was used as a measure of the in vitro effect of immunotoxins. Briefly , cel l s (8x10 5 ) were incubated for 30 to 60 minutes with varying dilutions of the immunotoxin or ricin in phosphate buffer with 200 mM lactose and then washed three times to remove any unbound material. The cel l pel let was resuspended in leucine free RPMI-1640 medium and plated out onto a 96 well flat bottom microtitre plate in triplicate. The cel ls were then pulsed with [3 H ] leucine (1muCi/well) and incubated at 37°C for 16 to 24 hours. The cells were harvested onto glass fiber filters and the incorporated radioacti v ity was counted with a scintil lation counter. The results were expressed as the percentage inhibition of [3 H ]leucine incorporation relative to control cells which received PBS as treatment. I nhibition of χ. £ t £ x i c it^ : In this assay the cytotoxicity of the immunotoxin was inhibited by free MoAb. Ce l l s (8x10 ) were treated with a constant amount of immunotoxin (220 ng/ml) and 2 different MoAb were then added in serial dilutions (anti-Ly-2.1 reactive and anti-Ly-1.1 n o n r r e a c t i v e ) . The cel ls were incubated for 1 hour and a cytotoxicity assay performed; the results were expressed as the percentage inhibition of cytotoxicity. RESULTS The study was conducted in 3 phases: (a) establishing - 86 - G optimum conditions for the coupling of ricin to MoAb; (b) characterization of the immunotoxin, including its binding to affinity matrices and SDS gel ana lysis; and (c) testing the potency of the immunotoxin on cell lines in vitro. ££M£ii£2 £_£ £i£i£ ££ iϋii H^i Ricin was chemically linked to MoAb via a disulfide bridge with the use of two bifunctioπal crosslinkers as described - the reaction of ricin with a 10-fold excess of SPDP introduced 2 to 3 pyridyl dithio groups based on the re l ease of 2 - r i d in e h i o n e (24), as shown in Table 81. Similarly, treatment of MoAb with a 5-fold excess of SAMSA introduced 2 to 3 protected sulfhydryl groups determined by reactions with dithiobis-2-πitrobεnzoic acid (Table B2) af ter deprotection with h dr ox 1 a ine (25). Subsequent reaction of the thiol groups of ricin yielded a ricin-antibody conjugate. The coupling method using SAMSA has several a d v a n't ages; it precludes the use of thiol reagents far deprotection and therefore prevents unnecessary dilution or removal of excess reagents, and it reduced the oxidation of free sulfhydryl groups and does not require low pH buffers. Of the ricin initially used, 70? was recovered in solution, 60? of this was bound to MoAb, 35 ? in the 210 KD complex (1 ricin molecule bound to 1 MoAb molecule), 25? in larger aggregates (2:1 , 3:1 , ..) and 40? remained unconjugated (Table 83), These yields were superior to the second method used in which MoAb was thiolated with SPDP. As shown in Table B3, the final yield of conjugated ricin was relatively low, being 10-15? of the ricin used initially. Conjugates were examined by SDS gel e 1 e c t r o p h o r e s i s . As found therefrom a 5? nonreduced gel indicated the immunotoxin consisted of unconjugated MoAb (150 KD) and a 210 KD complex (in a 1:1 molar ratio, determined by optical density measurements- and radioactivity). The au t oradiograph illustrated the 210 KD band to be radioactive and therefore indicated a complex that had one ricin molecule bound to one MoAb molecule. Additionally, under reducing conditions the immunotoxin was observed to consist of the heavy and light chains of immunog 1 obu 1 in (50 KD and 25 KD res ectivel ) and - B 7 - O s the expected 34, 32, and 30 KD subunits of ricin. £ill£i££2 §iS-____£ __11______.__. 2l -_-l---__12__£__j-Il£ i-l-- £__£-__l The galactose binding property of a whole ricin-anti-Ly-2.1 conjugate and SPDP thiolated ricin were first assessed on a Lactose-S epharose column (2 ml) by affinity chroma ography based on the affinity of ricin for sepharose. As shown in Figure B4, when thiolated unconjugated ricin was passed down a L a c t o s e -S e p h a r o s e column, ricin bound strongly to the column through its galactose receptors. Less than 5? was eluted from the column with PBS and lactose (100 M) was required to recover 90? of the aterial. However, when the r ic i - an t i -L y - 2.1 conjugate was applied to the Lactose- Sepharose column 86-90? of the conjugate, as detected by the radioactivity on the ricin moiety, failed to bind or be significantly retarded by the affinity matrics, when the column was washed with 2 column volumes of phosphate buffer. An additionalθ? of bound protein was eluted with the same buffer containing 100 mM lactose. Similar results were obtained with 6 different MoAb conjugated to ricin and the results are summarized in Table 84. Antib£d_ _ A£ ivi^ _ of I!li. l£i£_J An ib£dy_ The antibody activity of thiolated MoAb was tested. Thiolation of the MoAb with SAMSA did not significantly effect its binding to target cells. Aπti-Ly-2.1 thiolated with a 12 fold excess of SAMSA per MoAb molecule. This did not significantly alter the binding capability of the MoAb to E2 reactive target cel ls in a competitive binding assay using r a d i o i o d i n a t e d anti-Ly-2.1. As shown in Figure B5, the thiolated MoAb had similar activity to unmodified MoAb and increasing the concentration of unmodified anti-Ly-2.1 or thiolated anti- Ly-2.1 had equal inhibitory effects on the binding of 125 j - labeled anti-Ly-2.1 to E2 target cells. The Ly-2.1 MoAb was reacted with various amounts of SPDP and the effect on antibody activity measured with the use of rosetting assay (26). SPDP thiolation had no effect on antibody binding activity unless a very high substitution level (<9.2 molecules SPDP/mo 1 ecu1 e anti-Ly-2.1 ) was used. It should be noted that only a 6 fold excess of SPDP was used f or the c onjuga tion procedure wh ich l e d to the incorporation of 2 to 3 molecules of SPDP per MoAb molecule. I______-___2 2.1 ££_2___i2£.__££ J_£ U ____£££ ^ πe antibody activity of the conjugated proteins was measured by the rosetting assay. The titers of antibody before and after conjugation were determined as the dilution at which 50? of the target cells demonstrated rosettes. As shown in Figure B6 the activites were comparable for both the unmodified anti-Ly-2.1 and r i c i n - a n t i - L y - 2.1 conjugate on E2 target cel ls, indicating that the coupling did not effect the antibody reactivity. However, it shoul d be noted that noncoup led MoAb in the r i c i π - a n t i - L y - 2.1 conjugate could partial ly account for the conjugate binding. The reactivity was also tested by using a second cell line, D1 , the E2 and D1 cell lines being high and low Ly-2 + variants respectively of the ITT(1 )75NS cel l line . Both these cel l lines were identica l with the excep tion of their Ly-2.1 antigen density. As shown in Figure B6, there is a difference in the reactivity of conjugates with the two cell lines and clearly the binding o f th e c onju g ate was d ependent on the concentration of reactiv e antigen binding sites on the target cells. No binding was found with a noπreactive riciπ- anti-Ly1.1 conjugate on the E2 cel l line (Ly-1.1 ~). These r es u lts indicated that wh o l e ric in was succes sf u l l y conjugated with the MoAb and that the conjugated ricin did not play a rol e in the binding activity of the conjugates. £_._.£__£>___£______ £J_ ____.£ ■_.__Ξ£££J_£__i.__£ " T e specificity of the ricin toxin, which normally binds,- enters, and kills cells through receptors containing galactose, has been altered by c o v a l ent l y binding it to a M oAb. H ybrid conjug a tes (immunoto ins) of ricin and MoAb were synthesized with the bifunctiαnal crossliπking agent SAMSA and SPDP and were shown by af f inity ch r omatography no t to bind to the galactose residues of L a c t o s e - S e p h a r o s e or S e p h a r o s e - 4 B . Furthermore, the reactivity of the immunotoxins was dictated by the specificity of the cov alently bound MoAb. It was, therefore, necessary to show that the inhibition ef fects produced were specific. Three procedures were performed. - B9 - (π * £i£££i_£-_£ ' The immunotoxins, free ricin and MoAb, were assayed for inhibition of protein synthesis in reactive target cells. The MoAb alone at concentrations as high as 10 mug/ml did not inhibit protein synthesis in the target cells (not shown). Figure B7 shows the d o s e - r e s o n s e of ricin and ricin- anti-TFR in CEM (TFR + ) cells. The anti-TFR immunotoxin on a molar basis, in the absence of lactose was as potent as free ricin (Figure B7a) and reduced the capacity of cel ls to incorporate ([ H ] leucine by 50? at 30 ng/ml. Lactose, which competitively blocks ricin binding to cells, inhibited the toxicity of ricin 40-fold in CEM cel ls. In contrast, the anti-TFR immunotoxin was inhibited less than 2-fold by lactose (Figure B7b). Thus, in the presence of 100 mM lactose, CEM cel ls were 40-fold more sensitive to the immunotoxin than ricin. These results were coπfimed in repeated experiments with other immunotoxins. For example, the cytotoxic activity of a ricin ( 10 4 "" /BH-.~ treated) - anti-Ly-2.1 conjugate was tested on reactive target cells. The conjugate inhibited protein synthesis in vitro as shown in Figure B8. Lactose inhibited the observed toxicity of ricin, at least 40-fold in E3 cells, however, the observed inhibition of the immunotoxin was not altered by the addition of lactose and 50? inhibition of [ - ^ ] leucine incorporation occurred at 65 ng/ml. £__£££__£__£ __ To determine whether the observ ed inhibition was due to specific binding of the MoAb, the cytotoxic activity of two immunotoxins, an antigen reactive ricin-aπti-Ly-2.1 immunotoxin and a noπreactive ricin-anti- Ly-1.1 immunotoxin was tested on E3 cel ls (Ly1 -,2 + ). As shown in Figure B9a, there was no significant loss of cytoxicity by linkage or ricin to the Ly-2.1 MoAb, in the absence of lactose. This immunotoxin reduced the capacity of cel ls to incorporate [ - ] leucine by 50? at 45 ng/ml, whereas, the nonreactive ricin-anti-Ly-1.1 immunotoxin was ineffective and showed a low toxic effect on the E3 cel ls, where a concentration as high as 1000 ng/ml was required to inhibit protein synthesis. Thus, in the absence of lactose - 81 Q - ( » the reactive immunotoxin was 20 times more effective at inhibiting protein synthesis in E3 cells than a nonreactive immunotoxin. When 100 M lactose was introduced into the assay (Figure B9b) the cytotoxicity of noπconjugatad ricin was inhibited (50? inhibition of [3 H ] leucine incorporation occurred at greater than 1000 ng/ml ) whereas the cytotoxicity of the anti-Ly-2.1 immunotoxin was not significantl altered. The nonreactive immunotoxin however, was not toxic to E3 cel ls at concen rations as high as 104 ng/ml. Thus, in the presence of lactose, E3 cells were more than 200-fold more sensitive to the reactive immunotoxin than the nonreactive immunotoxin. £££££______£ 1 Additional experiments were performed in which the cytotoxic activity of three immunotoxins was tested on reactive and nonreactive target cells. The immunotoxins were -able to inhibit protein synthesis in cultured cells bearing the appropriate target antigen. The MoAb after conjugation were found to maintain their specificity . The effect of an anti-TFR immunotoxin on [3„] leucine incorporation into CEM cells (TFR + ) and E3 cel ls (TFR " ) is shown in Figure B10a. Both call lines were equally inhibited by ricin in the absence of lactose (50? inhibition of protein synthesis occurred at 20 ng/ml). The anti-TFR immunotoxin resulted in greater than 50? inhibition of [3n] leucine- incorporation by CEM (reactive) cells at 6 ng/ml. lifhereas 80 times more immunotoxin was required to inhibit protein synthesis in the nonreactive E3 cells. Two different preparations of ricin-anti-Ly-2.1 demonstrated marked inhibition of protein synthesis in E3 (Ly-2.1 + ) cel ls. EL4 cells were more sensitive to ricin (I0 4 ~/BH 3 ~) treated than E3 cells (Figure 810c), whereas Bli/5147 cells were 3 times less sensitive to ricin (data not shown). EL4 and Blι! 5147 target cel ls that lacked the Ly-2.1 specificity were assayed for sensitivity to ricin-anti-Ly- 2.1. In Figure 810b and 810c the sensitivity of these cel l lines to the anti-Ly-2.1 immunotoxin in the absence of lactosa is shown. No inhibition of protein synthesis was •s. - B 1 1. - ( - "- . seen at the highest concen ration of the immunotoxin (>10Q0 ng/ml) tested, whereas the anti-Ly-2.1 immunotoxin showed marked inhibition protein synthesis in E3 (Ly-2.1 + ) cel ls and 50? inhibition of [3 H ] leucine incorporation of E3 cells occurred at 100 ng/ml (Figure B10b) and 60 ng/ml (Figure 10c) for the r i c i n - ant i -L y - 2.1 and ricin (NaI0 4 " /BH 4 ~ treated) -anti-Ly-2.1 preparations respectively. Thus, no a π t ib o d - me di a t e d toxicity of the anti-Ly-2.1 immunotoxin was not detected in Ly-2.1~ cells. J_Q_l___l_t-_i22 2.Ϊ. £__-_2-_2-___£___-_- The mechanism of the specific cytotoxic action of the immunotoxin relied in part on the MoAb moiety binding to the target cel ls via a cel l surface receptor. The specific cytotoxicity of a ricin (NaI0 4 ~/BH 4 ~ treated) - anti-Ly-2.1 conjugate on E3 cells was blocked by the presence of excess aπti-Ly-2.1 as shown in Figure B11. The simultaneous addition of a 20 fold excess of aπti-Ly-2.1 (10 ng/ml) with the immunotoxin caused a 50? reduction in the cytotoxicity of the anti-Ly-2.1 immunotoxin to E3 target cells. In contrast, excess anti-Ly-1.1 (an isotype control MoAb directed to an irrelevant antigen) did not alter the cytotoxicity of th.e anti-Ly-2.1 results indicate that the cytotoxicity of the immunotoxin is mediated via their antibody binding ability. DISCUSSION In this part the coupling of ricin to antibody is reported, the most important resul t obtained was the immunotoxins produced were devoid of galactose binding. The study concentrated on several different aspects: (a) the design of an efficient coupling method which resulted in high yields of immunotoxin; (b) characterisation of the immunotoxin; (c) determining the binding properties of the immunotoxin; and (d) measuring the biological properties of the covalently coupled ricin and antibody. The use of whole ricin as an antitumor agent has been quite restricted and for obvious reasons, as this toxin is capable of binding to all cells. Ne ertheless, a phase I clinical trial has been conducted with the use of nativ e ricin (27) and a t h e r a p e ut i c a 11 y safe dose was found to have only marginal - B12 - (**<>' antitumor ef f ects. The concept of cova l ent ly coup l ing the who l e toxin to tumor specif ic antibodies has b een extensively researched (28,29) and successful studies have shown that whole ricin immunotoxins are powerful reagents in vitro in the presence of lactose. Coupling procedures previously used for the cαvalent attachment of ricin to MoAb resulted in very poor yields of immunotoxin, presumably due to different reasons including the use of reducing agents to introduce free sul fhydryl groups into MoAb, low pH buf f ers which resulted in the precipitation of concentrated protein solutions and rapid o xid ation of f r ee su l f hydry l groups during coup l ing reaction. Thiolation of MoAb with SAMSA overcame a number of these problems, i.e. deprotection of the sulfhydryl groups of the MoAb and coupling to thiolated ricin is carried out in the same buffer as the coupling reaction. In this part, a number of parameters were carefully monitored, particul ar attention was paid to the number of activ e thio l groups introduced , th e mo l ar ratio of ricin and M oAb in the r eact ion mixture and the reaction time. The co v a l ent attachameπt of ricin to MoAb resul ted in up to 60? of the recovered ricin being conjugated, 35? of the ricin was present in a 21 KD species where one ricin molecule (Mr 65 ,000) was attached to one MoAb mol ecule (Mr 150,000) (Tabl e B3). Thio l ation of the MoAb with SAMSA with ricin with SPDP did not significan ly change the binding ability of th es e pro teins. Coup l in g reactions specif i c f or a particular amino acid residue could theoretically denature the antigen binding site of certain antibodies during coup ling or cou ld result in extensiv e recipitation of protein due to th e a l teration of it s ch arg e. Neither occurred to any extent with SAMSA, and up to 4 molecules of SAMSA coul d be incorporated per MoAb mol ecul e with the retention of 100? of the antibody activity (Table B2). SPDP thio l ate d ricin (2-3 SPDP r e s i d u e s / r i c i n mo l ecu l e ) maintained an active galactose binding site (Table 84). The blockage of the galactose binding site was a specific event and a direct consequence of the close proximity of the Fc - B 1 3 - - 2 - • portion of the MoAb sterical ly blocking the galactose binding sites on the ricin molecule. When whole ricin was covalently attached to MoAb and the purified immunotoxin (210 KD) passed down Lactose-Sepharosa affinity column, 90? of the immunotoxin failed to bind the galactose residues. Immunotoxins produced with whole IgG anti-Ly-2.1 (lgG2a) and anti-TFR (IgG1 ) were equal ly cytotoxic in the presence or absence of lactose (Figure B7-B9). The rationale for constructing an immunotoxin is that a cytotoxic agent coupled to a MoAb is both cytotoxic and selective in action. The success of such an approach depends in part on whether the toxin and antibody retain their biological activities after conjugation. A number of studies were then performed to demonstrate that the immunotoxin reacted specifially with target cells carrying the antigen to which the antibody was directed and did not react with tumor cell lines which lacked this antigen. The anti-Ly-2.1 immunotoxin was shown to be effective and specific for reactive cel ls in vitro. A dose of 200 ng/ml efficiently reduced the protein synthesis of E3 target cells by >90?, whereas a nonreactive immunotoxin was not cytotoxic to these cells at the same molar concentration. Specific cytotoxicity was shown to be dependant on the anti-Ly-2.1 immunotoxin binding to the cell surface receptors since cytotoxicity was only inhibited upon addition of excess anti-Ly-2.1 but not by the addition of an unreactive MoAb (Figure 811 ). In addition, an anti-TFR immunotoxin was shown to be 4 times more toxic to CEM, TFR positive target cel ls than free ricin, >90? inhibition of protein synthesis was achieved with 50 ng/ml of the immunotoxin without affecting receptor negative E3 cells. Futhermore, 90 times more immunotoxin was needed to inhibit the protein synthesis of the nonreactive E3 cells (Figure B10a). The selection of whole ricin for in vivo im unotherapy was based on the biological importance the B chain plays in the potentiatioπ of the A chains potent cytotoxicity (5-7). The advantage of ricin conjugated to MoAb is that the cytotoxic nature of the toxin can be focussed to the surface of a particular target cell resulting in the specific killing of the cell. The blockage of the galactose binding site was a fortuitous, but reproducible finding as more than eight immunotoxins have been produced with different MoAb which are similarly specifically toxic. There have been few studies performed with whole ricin immunoto ins, although a thioether linked immunotoxin was prepared with a blocked galactose binding site (30) and in vitro studies were repor ed. In this Part ricin was covalently attached to MoAb with the use of a disulfide bridge. The cytotoxicity of the immunotoxins was restricted to MoAb re a c t i e " c e 11 s and on the basis of the encouraging in vitro results the in vivo efficacy of the blocked immunotoxins was examined and reported in Parts C and D. REFERENCES Part B 10. HYMAN R, STALLINGS V. Complemen ation patterns of Thy- 1 variants and evidence that antigen loss variants "pre- exist" in the parental population. JNCI 1974; 54:429-436. 11. GH0SE T, GUCLU A, TAI J, et al. Immun oprophy 1 ax is and immuπotherapy of EL4 l ymphoma. Eur J Cancer 1977; 13:925-935. 12. S MYTH MJ, PIETERSZ GA, CLASS0N BJ , et al. The specific targeting of ch l orambuci l to tumors, JNCI 1985; 76:503-510. 13. F0LEY GE, LAZARUS H, FARBER S, et a l. Continuous culture of human lymphoblasts from peripheral blood of a child with acute leukemia. Cancer 1965; 18:522-529. 14. HOGARTH PM, HENNING MM, McKENZIE IFC. Monoclonal antibodies to murine Ly-2.1 cel l surface antigen. Immunology 1982; 45:135-144. 15. HOGARTH PM, POTTER TA, CORNELL FN, et al. Monoclonal antibodies to murine cell surface antigens. J Immunol 1980; 125:1618-1624. 16. PANACCI0 M. Monoclonal antibodies to the human transferrin receptor. BSc (Hons) report. Parkvil le, Victoria, Australia: Department of Pathology, University of Melbourne, 1983. - B15 - C O 1 17. THOMPSON CH, JONES SL, PIHL E, et al. Monoclonal 2 antibodies to human colon and colorectal carcinoma. Br J 3 Caner 1983; 47:595-605. 4 18. THOMPSON CH, JONES SL, - H I T E H E A D RH, t al. A human 5 breast i s s u e - a s s o c i a e d antigen detectetd by a monoclonal 5 antibodv. JNCI 1983: 70:409:419. 7 19, 8 9 10 21 11 12 mixtures. Effects on toxicity a 13 distribution. Eurr JJ.. BBiiααcchheem 11998855;; 114477::119977--220066.. 14 22. GREENWOOD FC, HUNTER 111 M , GLOVER JS. The preparation of 1 31 15 I - l abe l l ed human growth hormone o f high sp ecif ic 16 radioactivity. Bioche J 1963: 89:114:123. 17 24. CARLSSON J, DREVIN J, AXEN R. Protein thiolation and 18 reversibl e r o t e i n - p r o t e i n conjugation. Biochem J 1978; 19 173:723-737. 20 25. ELLMAN GL. Tissue sulfhydryl groups. Arch Biochem 21 Biophys 1959; 82:70. 22 2 266.. PPAARRIISSHH CCRR,, MMccKKEENNZZIIEE IIFFCC.. AA sseennssiittiivvee rroosseettttiinngg mmeetthhoodd 23 for detecting sub populations of l yymmpphhooccyytteess wwhhiicchh rre. act 24 l.l I h _a * ϊ . _ι __ι ___ 4 α α τ_ α _ T T mmn n n . M ύ KnidHse 119Q7.8Q;. 2_0n:.117*73"?-_118Q3.. 25 26 27 28 29 30 31 I Immmmuunnoottooxxiinnss:: aa nneeww aapppprrooaacch to cancer therapy. Science 32 1983; 219:644-650. 33 30. THORPE PE, ROSS ti/CJ, 8 R 0111 N ANF, et al. B lcokagte of the 34 galactose binding sites of ricin by it3 linkage to antibody. 35 Specif ic cytotoxic ef f ects of the conjugates. Eur J. 36 Biochem 1984; 140:63-71. - β/« - l 2 ^ Table β / (a) SPDP modification of MoAb Molar excess SPDP No. molecules SPDP/molecule MoAb anti-Ly-2.1 anti-Ly-1.1 2 0.2 5 1.8 6 2.8 10 2.5 12 4.1 18 7.2 24 9.2 50 11.1 (b) SPDP modification of Ricin 1 Molar excess SPDP No. molecules SPDP/molecule Ricin pH 7.0 pH 8.0 4 0.5 5 2.0 6 0.9 7.5 1.0 10 3.2 15 3.9 20 5.5 The number of SPDP molecules introduced per protein molecule was determined by calculating the liberation of pyridine-2-thione. Table β 2- SAMSA modification of MoAb 1 Molar excess SAMSA No. molecules SAMSA/molecule anti-Ly-2.1 5 2.9 10 4.3 The number of SPDP molecules introduced per protein molecule was determined by reactions with dithiobis-2-nitrobenzoic acid. - __. . Table & " b Comparison of the coupling efficiency of two different procedures used to covalently link ricin to MoAb. Z INITIAL RICIN 1 Procedure l2,3 Procedure lr (SAMSA/SPDP) (SPDP/SPDP) ricin-MoAb (1:1 . ) 29 25 13 ricin-MoAb (2:1,3:1,...) 12 17 8 unconjugated ricin 29 27 48 Recovery 70 69 69 1 The results are expressed as the Z initial ricin used. 2 See materials and methods for details. •* ' Two different conjugations are reported with procedure one to illustrate the reproducibility of this method. Table G*- (a) Binding affinity of SPDP modified ricin. X Initial ricin Lactose-Sepharose Sepharose-4B Ricin Modified Ricin 1 Ricin Bound 78 62 61 Not bound 5 2 9 Recovered 83 64 70 (b) Binding affinity of ricin-anti-Ly-2.1 on Lactose- Sepharose Z Initial ricin Lactose-Sepharose 22°C 4°C Bound 7 8 Not bound 77 86 Recovered 84 94 Ricin chemically treated with sodium metaperiodate and sodium cyanoborohydride. (c) Binding affinity of ricin-MoAb conjugates on Lactose- Sepharose Immunotoxin X Initial ricin Bound Not Bound Recovered anti-Ly-1.1 0.5 89 90 anti-TFR 23 77 100 anti-Ly-2.1 7 87 94 (modified ricin) 250-30.6 16 83 99 anti-HLA 7 75 82 17.1/2 6 80 86 - C1 - -l 2 -^ £sii £ This part investigates the in vivo stability of ricin and ricin (I0 4 ~/BH 3 ~ treated) and evaluates the use of disulfide linked antibody conjugates for the treatment of tumors in vivo. The ability to successfully use whole ricin was due to the loss of the galactose binding sites on binding ricin to antibody to produce conjugates with limited nonspecific binding. MATERIALS AND METHODS J_J.££ (5S8L/6 x BALB/c)F1 (B6CF1) mice were produced in the Department of Pathology, University of Melbourne. _.£]_££ £ii£ radiation induced murine thymoma ITT(1)75NS E3 (8) was grown in 10? newborn calf serum in DMEM. The human T-cell leukaemia line CEM (9) was maintained in RPMI-1640 with the same additives. The E3 cell line was maintained by serial passage into the peritoneum of 86CF1 mice. For in vivo experiments the cells from the ascitic fluid were washed and centrifuged (1,500 rpm for 5 minutes) twice in DMEM and PBS, resuspended in PBS, and injected s bcutaπeous 1 y or in traper i t on ea 11 y into B6CF1 mice. £££j_.£_l_£i_.__a ji £ __£diJ_A££_iA£_2 £JL Ei£_kl_ H___ii2 I0 4 ~/CNBH 3 ~ £_!£_■££££ The method of carbohydrate modification has been described in detail in Part B. Extensive details of the characterization of ricin modified by I0 4 ~/CNBH 3 ~ treatment have been published elsewhere (7) but to briefly summarize, the properties were as follows. A slight (<500 D) reduction in molecular mass of both A and B chains was seen when ricin treated with I 0^-, ~/CNBH 3 ~ was analyzed on SDS po 1 y aery 1 amide gel e 1 e c t r o h o r e s i s in the presence of 2 - e r c a t o e t h a n o 1. This may have been due to the elimination of CH.OH from carbohydrate residues. There was no significant change in the amiπo acid composition of ricin after treatment. No splitting of the disulfide bond between the A and B chains was detectable when modified ricin was analysed on SDS gels in the absence of 2-mercaptoethanol. There was no significant loss in the ribosome-damagiπg e ' ffect of the A- chain moiety of the modified toxin. Similarly, the carbohydrate-binding property of the B-chain was fully - C2 - (2.?) preserved, as also shown in Part B by its Sepharose-binding ability. The extent of the carbohydrate modification depended on the duration of the treatment, and resulted in the maximum destruction of 70? of the mannose residues and all xylose and fucose residues. In i.-_-_£ £i£i£i_2-___-k£_! £__ _.£.__.____£ ££__ __£__.____.__£__ £i£A_2 ( IO A " /CNBH " treated) This study compared the iodistribution of ricin and I0 4 " /CNBH 3 ~ treated ricin in B6CF1 mice. Ricin (50 mug, 1 mg/ml) or modified ricin (I0 4 ~ /CNBH 3 " treated) was labeled in the presence of 100 mM lactose with ' 2 ~ I using ch 1 or a ine-T. Groups of 3 mice were sacrificed 30 minutes after the injection of labeled toxin. Each mouse was given an injection in the tail vein of 0.12 mug of the toxin (Specific a cti vity =_ 4.6-5.8x106 cpm/mug). The b iαdistr ibut ion of 1 M25 abel was determined by counting the radioactivity of blood, liver, spleen, kidney and heart from these mice. Organs were weighed and the distribution of isotope measured in a gamma counter. Blood activity was calculated on the assumption that the overall blood volume represents 7? of the total body weight. Organ distributions were then calculated as a percentage of the original injected dose found in the organ. ££££££ .____.££ £j_ £i£i£__£££.i__£___{ ££__i__22__££ Ricin or modified ricin (I0 4 ~/CNBH 3 ~ treated) was conjugated to MoAb by a disulfide bridge using two bifunctional crossliπking reagents, SAMSA and SPDP, according to the method previousl described in Part B. The resultant ricin-antibody conjugates had a diminished galactose binding capacity. The native ricin-antibod conjugates were further purified from unconjugated MoAb by affinity chroma ography on a column of Concana a1 in-A immobilized to Sepharose (10). According to this method unconjugated MoAb, which did not bind the lectin, was discarded in the filtrate while ricin because of its mannose containing o 1 igosaccharide, binds to Con-A. Thus only MoAb bound to ricin is retained on the affinity column. The immunotoxin was eluted in a solution of 0.5 M methy1-a lpha-D-mannoside. 5!__.._.£_k.____£___.£2 2l £._.£._..__._. Efidili≤^ ____2_-- __£__ £__£_-__z i _ , HΪΪ-. o d _\ ££___i__2£-_££ Ricin, modified ricin (I0 4 " /CNBH 3 ~ 2 treated) and ricin-MoAb (purified on C oπ -A - S e p h a r α s e to 3 remove unconjugated MoAb) were labeled using the iodobead 4 method: ' one iodobead (Pierce Chemical Company, Rockford, 5 Il linois, USA) and 200 mul of proteπ (approx. 10 mug) were 6 al lowed to stand at room temperaure for 5 minutes and 2.5 7 mCi of carrier-free Na * I (Amersham International Ltd, 8 Amersham, England) then added and the reaction monitored for 9 a further 5-15 minutes. The iodinated protein was separated 0 from free iodine by gel filtration using a PD-10 Sephadex ,1 column. 2 An.tjLbody. activity , a n d sgeci f ici_ty. f 1 2S I-labelβd __ £A£______££_-___.££___ ££_Q.____.2£___££ τ π *-- i m unor e a c t i v i t y of the 4 radiαlabeled immunotoxin preparations was measured by a 5 direct in vitro cell binding assay. The binding ability of 125 6 I-labeled riciπ-aπti-Ly-2.1 was tested on two different 7 target cell lines, one being reactive with the immunotoxin, 8 the other nonreactive. Cells used for the assay were E3 (Ly- 9 2.1 + ) and CEM (Ly-2.1 " ). Cells (1x10 δ ) were added to tubes 0 pre-blocked with 1 ? BSA in PBS and the 1 25 I - 1 a b e 1 e d 1 immunotoxin was added at doubling dilutions (in the presence 2 or absence of 200 M lactose) and the tubes were incubated 3 on ice for one hours. The cells were then washed with PBS or 4 PBS containing 100 mM l actose (4x5 mis), and the cel l 5 pellets counted for one minute in a gamma counter. 6 ££__.££ .__.__A££ ££££.__ I π this assay the binding of I - 7 labeled r ic in- a n t i- - 2.1 to E3 reactive target cel ls was 8 inhibited by free MoAb. The labeled immunotoxin had a 9 specific activity of 3x10 cpm/mug. The association of 0 iodinated immunotoxin with cel ls was examined. E3 cel ls 1 (3x10 s ) were added to tubes pre-blocked with 1 ? BSA in PBS 2 and 1 25 I-labeled r i ci n- an t i- y- 2.1 (13 ng/ml) and various 3 dilutions of unlabeled anti-Ly-2.1 or anti-Ly-1.1 were 4 added. The cells were incubated on ice for 30 minutes and 5 then washed 4 times with PBS to remove any unbound material. 6 The cel l pellets were counted for one minute in a gamma 7 counter and the results were expressed as the percent 8 inhibition of the maximum counts bound. - C4 - lfl ' £A££1£ £i££££H££ £_£ £AHi S£ " A£ £^ £ £A£ (10 " /CN8H ~ - ££HJH2£l££ 1 " - 1 β following 3 preparations of ricin were labeled with ' z 5 1 using an 4 iodobead to a specific activity of 1.2 - 1.7 x 10' cp /mu g: 5 (a) untreated ricin; (b) ricin treated with Iθ ~/C BH 3 ~? (c) 6 r icin-an ti-Ly-2.1 purified by affinity chromatography on a 7 Con- A-S epha ro s e column. Groups of 4 female 86CF1 mice were 8 injected with the iodinated protein solutions via the tail 9 vein. Each mouse received either 0.24 - 0.3 mug of the 0 toxins or 0.81 mug of the immunotoxin in 100 - 150 mul PBS. 1 Blood samples were collected at timed intervals in heparin- 2 treated tubes and plasma mas separated by c e n t r i f u g a t i on. 3 The time-course plasma level of the radiolabeled protein was 4 then evaluated and the results expressed as a percentage of 5 the initial dose injected. Blood activity was calculated on 6 the assumption that the overall blood volume represents 7? 7 of the total body weight. 8 Serum samples (5 ml) were further analyzed by SDS-gel 9 e 1 ectr ophor esis using 5? and 12.5? acryla ide gels in order 0 to determine the stability of the radiolabeled proteins in 1 vivo . 2 J.rι v^Ji£ £££££]_£_,._ £,£ 1_J£A__£ _jH_-2££ *-• n a survival study, 3 tumor cells were grown in the peritoneum of mice in the 4 ascites form and treatment .as delivered into the ascites 5 fluid. The percentage survival of mice in each group was 6 plotted as a function of time. In a tumor growth study, 7 tumor cells were injected subcu t aneous 1 y into the abdominal 8 wall of the mouse and allowed to develop into palpable 9 tumors measuring 0.5 - 0.8 cm in diameter before treatment 0 was commenced. Mice were then subjected to a series of 1 intravenous treatments, and the size of the tumors measured 2 daily with a calipβr square. Measurements were made along 3 the erpendicular axes of the tumors, and the data was 4 recorded as mean tumo size (product of two diameters ± 5 standard error of the mean). Experimental groups of 10 - 40 6 mice of the same sex were used in each experiment. 7 l£iA£A£_l i_£ SA££ Ricin and ricin-antibody conjugate 8 solutions in PBS were injected intraperitoneal ly or intravenousl into groups of three to five male BSCF1 mice. The LD50 was calculated according to lilell (11). ϋA2_-2E2_---£--£2-- BSCF1 mice weighing 25 g and 30 g were injected intra eritoneally with ricin (3.3 mug/kg body weight) or ricin that had been treated with sodium me aperiodate and sodium c y a π ob o r o h y d r i d e or of ricin- antibody conjugates (3.3 mug ricin/kg). The animals were sacrificed 1 to 2 days later and the following organs were removed and fixed in 10? formalin: liver, spleen, pancreas, kidney, heart and lung. The organs were sectioned and stained with haematoxylin and eosiπ far light microscopy. RESULTS The objectives of this study were firstly to examine the plasma clearance of ricin after intravenous injection and secondly to assess the in vivo efficacy of the ricin- antibody conjugates sythesized with reduced capacity to bind to galactαse. In ^ _2 bjLoJl £££A-l-_AA22 2£ £A2Aj_ ££__ 2!£^A£ £^ £ £AH (I0 4 " /CNBH 3 ~ treated^ The b io d is r i b u t i o n of 1 2 S I - 1 ab e 1 e d ricin and modified ricin was examined by injecting mice in ravenously with the toxins and determining the relative amounts of radiolabel accumulated in the tissues. As shown in Table C1 , ricin was rapidly cleared from the bloodstream and only 9? of the toxin remained in the blood 30 minutes after injection. The liver was the major site αf clearance and 36? of the injected ricin was trapped by the liver. The radioactivit per gram of liver was 6 times greater than in the blood. Ricin also accumulated in the spleen as shown by the greater levels of radioacti ity per gram of tissue (24.4? injected dose/g). The kidneys and the heart contained smal l amounts of radioac i ity. The kidneys had twice the amount found in the blood per gram, whereas the heart had a similar level. Ricin that had been treated with me a eriodate and cy anoborohydride was removed much less efficiently than the native ricin by the liver and only 8? of the modified toxin was found in the liver 30 minutes after injection, 4 to 5 times less than the observed level of native ricin (per gram - C6 - (ii) of tissue). 22? of the modified toxin was still present in the blood (Table C1 ), twice the level of native ricin. The spleen, kidney and heart all took up the modified ricin to the same extent as native ricin. Thus the modified ricin ( I0 Λ " /CNBH 3 " treated) had a greater in vivo blood circulation life than native ricin. The carbohydrate portion of native ricin is recognized by the liver and spleen and is thereby cleared f rom the bloodstream by the hepatic r et icul oendothe 1 ia 1 system. Specificity of 125 I-labeled r icin-an i-L y- 2.1 The 125 I- labeled ricin-an t i-Ly-2.1 conjugate was tested in different serological assayβ to determine whether the labeling procedure damaged or altered the binding or specificity of the MoAb. Binding of 125 I- 1 abe 1 ed-anti-Ly-2.1 to antigen-posti v e £3 cells and antigen-nega ive CEM cells in the presence or absence of lactose is shown in Figure C1 a. Clearly, there wa3 a major difference in the binding of the labeled immunotoxin to the reactive cells (E3) when compared to that of the nonreactive cells (CEM). The reactive cells bound up to 10 times more immunotoxin than did the nonreactive cells, also the binding of the immunotoxin to reactive cel ls was slightly inhibitied with the introduction of lactose (Figure C1b). The significance of this reactivity was demonstrated when the labeled conjugates were analyzed by SOS-PAGE using 5? acrylamide gels. It was shown in two different ' 25 I - labeled r i c i n - a n t i -L y- 2.1 preparations, one labeled using Ch1 oramine-T and the other using an iodobead, there is free antibody (or heavy chain di er) and unconjugated ricin present. Inte estingly, both labeling procedures resulted in some breakdown of the immunotoxin. Thus the free ricin in the preparation could account for the nonspecific uptake observed. To ensure that the binding of the immunotoxin to target cells was specific, after labeling had occurred at the antibody binding site, studies were performed to inhibit the binding of conjugate by free MoAb. As shown in Figure C3, the binding of 1 5 I-labeled r i c i n - a n i - L y - 2.1 could be - C7 - ( reduced by 50? upon the addition of 60 mug/m of aπti-Ly-2.1 , whereas a noreactive isotype control MoAb Ly-1.1 did not inhibit the binding even at MoAb concentrations as high as 200 mug/ml. Clearly indicating that the binding of the immunotoxin is directly related t its a n t i b o d y- b i π d i n g ability. £A££J2£ £l£££££££ °1 £A£ £i E£__.A__.A£__ £A£A____ ££!_! £A£Aϋ_l -,!l-.-.-,9._.l ££H_lM2li££ All 5lA££ Plasma concentrations of '"I- labeled ricin, modified ricin and ricin-antibody conjugates following intravenous injection in mice were determined at various times. Results were expressed as a percentage of the initial dose injected and plotted against time. As shown in Figure C4, native ricin displayed an extremely short plasma "survival" time. Ul i t h i π 30 minutes, more than 90? of the initial dose was removed. Contrary to what was observed with native ricin, modified ricin treated with me aperiodate and c a n o b o r o h y d r d e and ricin-antibody conjugates remained in circulation for a much longer period of time. After an initial decline, 30 to 40? of the initial dose was present in the blood 30 minutes after injection and up to 20? of the initial injected dose was in circulation 4 hours after injection (Figure C4). The same plasma samples taken at the various time interv a l s ere f urther ana lyzed by SDS-PAGE gel e 1 ec t rophoresis using 5? and 12.5? acrylamide gels. Results were that ricin was cleared quickly from the plasma, and there was only trace amounts of the toxin detected at 2 hour s an d there wa s no detectab l e radioactiv ity corresponding to ricin by 8 hours. In contrast, modified ricin was cleared much slower and could be detected intact in the serum 8 hours after injection. Ricin-an ibod conjugates were also detected in the serum samples. T he serum samples showed two major bands with estimated molecular weights of 150,000 and 200,000. Two other bands could also be seen on the gels of estimated Mr 65,000 and 110,000. The molecular weights of these bands estimated by their electrophoretic mobility in the gels, were close to valueβ calculated for unconjugated MoAb (Mr *_ 150,000), - C8 - ( .5 ricin-antibody conjugate (one MoAb linked to one ricin molecule, Mr - 210,000) and free ricin (Mr = 55,000). The fourth band detected was shown to be the heavy chain dimer of the MoAb (Mr -« 110,000) which was a byproduct of the labeling reaction. There was progressive loss of the band corresponding to ricin-antibo y conjugate (Mr -« 210,000), however, trace amounts could still be detected in the serum at 26 hours after injection. The relative amounts of protein in the bands of the autoradiograph culd be estimated by gel scanning. It has been often hypothesized that the disulfide bridge binding ricin to MoAb could be unstable in vivo due to th io- di su 1 fi de exchange. This could explain why plasma ricin-antibody conjugates disappeared quickly. If such a process was occurring, deconjugated MoAb would be generated rogressively in the circulation. Since MoAb molecules remain in the circulation a very long time, their presence should be easily measurable. SOS-gel e 1 e c t rop h 0 r e s i s revealed unconjugated MoAb and ricin-antibody conjugate at 30 minutes after injection, and progressive loss of the immunotoxin was observed. Contrary to what was predicted, the experimental results showed that plasma immunotoxin elimination was not associated with a increase of free MoAb, suggesting that it is very likely that splitting of the disulfide linkage does not occur in the blood. n vivo S.L L-.-.S.1 2.Ϊ. _,-,__ i_.A£A£z££.__A__£-__- £££____££-_££ The unique specificity of the ricin-an ibody conjugate, without the need to have lactose present, led to the possibility of using the conjugate in vivo. Al l experiments were performed with the E3 variant of the murine thymoma ITT(1 )75NS which in vivo is a rapidly prol iferating tumor with a doub l ing time of lass than 24 hours. Conjugates were tested in two types of tumor model. §M£)_A_-£A -,-.- l - n - n e f irst mode l B6CF1 mice wera injected intraperitoneally with 1-2 x 10 5 E3 cel ls and 6 - 24 hours l ater given one of the f ol lowing treatments in traperi tonea 1 y : (a) PBS ; (b) r i c i n - a n t i - L y - 2.1 (reactiv e conjugate)! ( <_ ) r i c i n - a π t i - L y - 1.1 (nonreactive conjugate); - eg- - (3«) or (d) a mixture of ricin and antibody. Al l treatments contained equivalent amounts of ricin and antibody which were 0.05 mug and 0.22 mug respec i ely. Three independent s udies were performed, the difference being the initial tumor cel l number inoculated, and the total amount of treatment received. The surviva l of these mice were monitored. £_£E££A_ Ϊ 2_!_ . 1*- Groups of 10 female B6CF1 mice (5 to 6 weeks old) were given intraper i onea 1 injections of 1 x 10 tumor cells and 24 hours later given one of the treatments described abo v e. Each reagent wa s administered intraperi oneally in 100 mul PBS. As shown in Figure C6a, mice that received a mixture of ricin and antibody died due to ricin toxicity by day 52 where 50? of the group had died by day 28, while groups receiving the nonreactive conjugate died by day 55 due to tumor similar to the PBS controls. The mice receiving the specific conjugate had a 90? survival and remained tumor free for greater than 200 days. £_SE££A_-£-_A 2: Groups of 10 female 86CF1 mice were inoculated with 2 x 10 5 tumor cells and 6 hours later give one of the following treatments; (a) PBS; (b) anti-Ly-2.1 ; (c) ricin-anti-Ly-2.1 ; (d) ric In-an i- 1 y-1.1 ; or (e) native ricin. The PBS treated mice had a survival time of 28 days, whereas the group that received either r i c i n - a n t i - L y- 1.1 , ricin or MoAb alone died within 36 days (Figure C6b). By contrast, 50? of the mice treated with r i c i n - a n t i - L y - .1 survived tumor free for more than 200 days. ££E££A_Q££i - : Groups of 20 to 40 B6CF1 mice were inoculated with 2 x 10^ tumor cells and 6 hours later the mice received one of the treatments mentioned above. Each reagent was administered intraperitoneall in 100 mul of PBS on days 0, 1 and 2 after tumor inoculation. The total amount of ricin and antibody given was 0.15 mug and 0.6S mug respectively. Figure C6c shows a highly significant antitumor effect of the ricin-an i-Ly-2.1 treated mice, and 25 out of the 40 mice survived on day 70, whereas ricin- anti-Ly-1.1 and P8S control mice all died by day 38. Mice treated with unconjugated ricin were all dead by day 25 - cιo - CV) . whereas mice treated with MoAb alone had some therapeutic 2 effect and mice were all dead by 52 days. ^ ____-!££ £££__-_-! __-_.M---- π * πβ second model conjugates were 4 tested for their ability to inhibit the growth of 5 subcutaneous tumors. Mice injected subcutaneous 1 y with 4 x 6 10 cells and on day 8 when tumors were 0.6 cm in diameter, 7 groups of 10 female 86CF1 mice were treated intravenously as 8 above. A further treatment was given on day 10, the total 9 doss being 0.2 mug and 0.9 mug for ricin and antibody 0 res ectively. On measuring the mean tumor size, on day 18, 1 the specific anti-Ly-2.1 conjugate treated mice had tumor 2 only 30? the size of the control group tumors (Figure C7). 3 The other groups had a similar tumor size to the control. 4 These mice were sacrificed and tissues were sectioned and 5 stained for histological examination. Mice in both 6 intravenous and conjugate treated groups showed extensive 7 damage to the liver, spleen and kidney, demonstrating their 8 nonspecific uptake by the ret ic u 1 oend ot he 1 ia 1 system in 9 these organs. 0 n vivo £f.f££t of modified £A£A____2____A-_22'-_ £££iH2£__££ 1 liiith the aim of specifically preventing loss of conjugate to 2 liver and other organs, ricin was treated with a sodium 3 emtaper ioda te and sodium cy anoba r ohy dr ide mixture in order 4 to chemically cleave the carbohydrate residues responsible 5 for hepatic uptake. The in vivo efficacy of the modified 6 ricin conjugates was performed using the tumor growth model. 7 Groups of 10 86CF1 mice injected s ubcut an eous 1 y with 1 - 3 x 8 10° tumor cells developed a solid tumor 0.5 cm in size 5 9 days after inoculation. Mice with established tumors 0 received an intravenous injection in 100 mul of PBS oft (a) 1 PBS; (b) modified ricin (I0 4 " /CNBH 3 " treated); (c) anti-Ly- 2 2.1 ; or (d) modified r i c i n - a n i - L y - .1 conjugate. Two 3 independent studies were performed the major difference 4 being the initial size of the tumors treated and the doses 5 given . 6 £._ . £££ ϋ_£-_i I s In - n -~ study mice were treated on days 5 7 and 8 with one of the treatments described above. The total 8 dose of modified ricin and MoAb received per mouse was 0.2 - en - _ 1 mug and 0.90 mug respectively. The tumor growth was 2 inhibited in mice that received the modif ied-ricin-ant ibody 3 conjugate in their treatment compared to the tumor growth 4 seen in mice that had been given PBS, modified ricin or 5 antibody alone. As shown in Figure C8a, by day 13 the mean 6 tumor size was only 70 to 75? that of the control mice, the 7 conjugate group having significantly smaller tumors than the 8 control groups. 9 £_SE££A__£____ _.'• '"'ice bearing smaller thymomas (0.3 cm 2 in 10 size) were treated on days 4 and 7 with one of the 11 treatments described previously. The total amount of 12 modified ricin and antibody given was increased to 0.25 mug 13 and 1.1 mug respectively. The results shown in Figure C8b 14 established significant ihibitioπ of tumour proliferation in 15 the group treted with modified ricin-an i-Ly-2.1 and by day 16 17 the mean tumor size was ap roximatel 60? that of the 17 controls. 18 Thus the modified ricin-anti ody conjugate had some 19 effect on tumor growth in the group with solid tumors. 20 The mice were sacrificed and tissues were sectioned and 21 stained for histological examination. In sharp contrast to 22 mice treated with native ricin, mice treated with the 23 modified ricin had only minor histological changes observed 24 in the organs. In particular the liver was normal and there 25 was little or no damage to the spleen. In erestingly, 26 however, the mice treated with the modified ric in-conjuga e 27 showed extensive damage to the liver and spleen. 28 £E__£££A££J_. £__ £EJ-A_i-_-_££ £.£__££__£ £__ £A£AH £Ei_ _2£i_A.fA£__ __£ £A£Aπ__£π_-A__££ " __ ££E_-E2£-_££ The antitumor effects of 3 30 different anti-Ly-2.1 immunotoxins were compared in a tumor 31 growth experiment. Groups of 20 B6CF1 mice bearing 32 established tumor grafts received an intravenous injection 33 in 100 mul of P8S of: (a) P8S; (b) anti-Ly-2.1 ; (c) riciπ- 34 anti-Ly-2.1 ; (d) r ic in- a nti-L y -2.1 F(ab)2; (β) modified 35 r ic i n - a n t i- y- 2.1 ; or (f) r ic in- 17.1 / 2. Mice were treated 36 in ravenously on days 5 and 7. The total dose of ricin and 37 antibody injected was 0.3 mug and 1.3 mug respec i ely. 38 The antitumor effects of the anti-Ly-2.1 conjugates were specific since antibody alone, ricin alone, antibody p lus ricin and nonreactive ricin-17.1 /2 control, did not significantly inhibit tumor, growth. As shown In Figure C9 r i c i π-a π t i-L y -2.1 F(ab')2 was a less effective antitumor agent than the two conjugates made with intact anti-Ly-2.1. The average tumor size of the animals treated with the F ( a b ' ) 2 conjugate was 65 to 75? that of the untreated controls. R i c i n- a n t i- L y - 2.1 and modified ricin-anti-Ly-2.1 were both effective antitumor agents. The protective effect given by the two aπti-Ly-2.1 conjugates was similar and the mean tumor size of mice treated with ricin or modified r icin-anti-Ly-2.1 was significantly reduced to 50 to 55? the size of the untreated controls. Thus as antitumor agents whole ricin anti-Ly-2.1 MoAb conjugates with either ricin or modified ricin were 20 to 30 times more effective at inhibiting tumor growth than the F(ab')2 conjugate and about 60 to 80 times more effective than the nonreactive ricin-17.1/2 control. l£__A£A____ _*£ UA££ Table C2 summarizes the results of the biological tests on ricin, modified ricin and antibody conjugates. Each set of results is representative of those obtained in two or more experiments. Ricin had an acute LDςn by either the i n t ra p e r i t on e a 1 or intravenous route of 1.7 mug ricin per kg body weight (mug/kg) an 0.8 mug/kg respecti ely. By contast, ricin coupled to immunog 1 obu 1 in was 4-fold to 5-fold less toxic to mice. The LDcn for ricin-antibody conjugates by the i ntraperitonea 1 and intravenous route was 7.0 mug/kg and 4.5 mug/kg respectively. The toxicity of ricin to mice was enhanced by treatment with the I0 * ~/CNBH 3 ~ mixture 2-fold by the intravenous route and the LDgg for the modified toxin was 0.4 mug/kg. However, mo d i f i e d - r i c i n - a n t i b o d y conjugates were 2-fold to 3-fold less toxic to mice compared to the ricin-antibody conjugates and the LD SQ for the modified ricin immunotoxins was 8.3 mug/kg. A£-_£E£i-l£A£2-- n -i- ~ histological changes in the organs of mice poisoned with native ricin and ricin-antibody - C13 - £ _ *• «>) conjugates were seen in the liver and the lymphoid tissue. They were essentially the same as observed by Dereπziπi et al (12). The liver, spleen and kidneys of mice that had received near-lethal doses of ricin were pale in color. The liver was congested with blood. Extensive damage was seen to the cells lining the siπusoides: apart from the occasional intact sinusoidal cell, there remained only pyknotic nuclei and nuclei debris. No damage to hepatic parenchymal cel ls was apparent. The spleen was also heavily congested with b lood. The follicles and germinal centres appeared to be intact but the red pulp was largely necrotic with non- pyknotic cells visible. All other organs appeared normal. In sharp contrast, only minor histological changes were seen in the organs of mice poisoned with ricin that had been m o d i f i e d b y t r e a t m e n t w i t h m e t a p e r i o d a t e a n d c y a n obo ro h yd r i de. In particular, the liver was normal and there was little or no damage evident to the spleen. However, mice injected intravenously with modified ricin- antibody conjugates showed extensive damage to both the liver and spleen. DISCUSSION In this part the use of whole ricin-antibody conjugates in vivo uas reported and the data presented indicated that, under certain circums ances, whole ricin-antibody conjugates can lead to complete eradication of tumors without the usual side effects associated with whole ricin. The previous use of whole ricin has been quite restricted and for obvious reasons this toxin is capable of binding to all cells. Nonetheless, a phase I study has bee conducted in patients using ricin alone, i.e. in the absence of antiody, and some effects noted (13). The covalent linkage of ricin to MoAb yielded an immunotoxin devoid of galactose binding, that was specificaly cytotoxic to MoAb reactive cells. On the basis of the encouraging in vitro results reported in Part B, in vivo experiments were performed and lead to some promising results; however, the success depended on the route of administration of the whole ricin-antibod conjugate. - C14 - t+ Firstly, using the in traperitonea 1 route with tumors growing in the ascites f orm there was complete eradication of the tumor (Figure C6). Whil e these results are encouraging, a scites tumors growing in mice were not considered to be equivalent to ascites found in patients where there are f requent l y l arge deposit s o f s o l id tumor s cattered throughout the peritoneum rather than just tumor cel l s in the f luid. Nonetheless, in pleural, pericardial or ascites f luid one woul d expect , on the basis of results shown h erein, whale ricin-an i ody conjugates to eradicate free tumor cells in suspension. Secondly, intravenous injection of whole ricin-antibody conjugate was used with some success (Figure C7). However , two problems are noted: firstly the conjugates given in ra venously do have some nonspecif ic toxicity on liver and spleen - presumably as they are taken up by r e ic u 1 αen d o t h e 1 ia 1 cel ls, possibly due to the high mannos e conten t of th e ricin . Th e second p rob l em o f intra venous use was that the efficacy of the treatment was reduced. This coul d wel l ha v e been due to the clearance of the conjugate by the r e t i c u 1 o e n d o t h e 1 i a 1 system, but may a lso ha ve been due to the dif ficulty of large complexes entering tumors. To pursue this strategy the carbohydrate residues on the ricin molecule was chemically modified and the b iologica l surviva l time of the modified ricin was compared to ricin and ricin-antibody conjugates. Immunotoxin loca l ization on the tumor is an obv ious critica l step in order to achiev e any in vivo therapy. It is t here f oe n ecessary t hat t he i mmunotox in r ema ins i n circulation at a sufficiently high level for a long enough period of time to allow binding to the tumor cells to occur. Thus the examination of the in vivo circulation life of the toxin and immunotoxin may elucidate the reasons for the poor in vivo tumαricidal effect of the immunotoxin. In this study t he biol ogical l ife time of ricin and modif ied ricin were evaluated. 1 I - 1 abe 1 ed ricin injected into mice was found to be removed rapidly from the blood stream by the liver and s p l e e n . T h e c l e a r a n c e o f t h e t o x i n b y t h e r e icu 1 oendothe 1 ia 1 system is likely to be mediated through - C15 - (<f2.) recognition of the high mannose o 1 ig o s a c c h a r i d e s of the toxin (7). Destruction of the carbohydrate portion of ricin using a mixture of sodium metaperiodate and sodium c yanoborohydride r educed the c l earance b y t he reticu loeπdo the 1 ia 1 system. The modified toxin caused none of the h is top a tho 1 ogica 1 changes caused by the native toxin to the liver and spleen. Carbohydrate modification reduced the rate at which the toxin is cleared from the bloodstream so allowing It to persist for longer periods and at higher concentrations (Figure C4), therefore it was argued that immunotoxins prepared from ricin that have been modified in this way should have a better chance of gaining access to and killing their target cells. Part B established the specificity and efficacy of the modified ricin-antibody conjugates in vitro, and this study evaluated the in vivo activity of the modified ricin- antibody conjugates. In tumor growth experiments, therapy did not commence until tumors were well established (0.5 cm in size). Of the treatments ad inis ered, the modified r ic in - a π t i- y- 2.1 conjugate was the most effective tumor inhibitor (Figure C8). The Ly-2.1 MoAb alone and ' the modified ricin alone displayed no significant antitumor activity towards the murine thymoma E3. The ricin and modified ricin immunotoxins prepared with intact Ly-2.1 MoAb were significantly better as antitumor agents in vivo than an immunotoxin made with the F(ab')2 fragment of the Ly-2.1 MoAb (Figure C9). This difference could be because the Fc portion of the intact MoAb contributes to the effective blockage of the glactose- binding sites on the B-chain of the toxin. The blockage of the g a 1 a c t o s β- b i πd in g sitas was not uniform using the F(ab')2 fragment. As shown in Part B, most of the ricin- F(ab')2 conjugate molecules that did not bind the Lactose- Sepharose column were retarded in their motion down the column. Also the F(ab')2 immunotoxin that passed through the Lactose-Sepharose column was not entirely f ree from nonspecific toxicity. Another possible explanation for the weaker antitumor activity of the F(ab')2 immunotoxin is that - C16 - (if* ) it is more rapidly cleared from the mice. Interes in ly this study showed that immunotoxins of ricin and modified ricin-anti-Ly-2.1 had an equal antitumor effect on the E3 thymoma in vivo, and tumor growth was inhibited by 50-55? (Figure C9). However, it should be noted that although the modified ricin had a 2-fold increased plasma circulation life, the covalent attachment of the native ricin to MoAb also increased the plasma survival time of the ricin molecule (Figure C4). furthermore, one must take into account that the ' E3 tumor divides extremely rapdily and perhaps a significant difference in antitumor activity between the two preparations could be observed with a slower growing tumor. An unexpected finding was that the modified ricin- antibody conjugate caused damage to the liver of mice treated intravenously. The toxicity could be due to binding to cells that have receptors for the Fc portion of the i munog 1 obu 1 in , however, liver and spleen damage was also inflicted by the F(ab')2 immunotoxin. From the data it is apparent that although there was no significant MoAb released after immunotoxin administration, i.e. the disulfide linkage connecting MoAb and the toxin was stable in the blood as demonstrated by the absence of increasing amounts of unconjugated MoAb in the circulation following immunotoxin injection. The conta ination of the immunotoxin preparation with free MoAb, even at a low level became a dominant feature in vivo after only a few hours due to the difference in elimination rates between immunotoxin and MoAb. For example only trace amounts of the immunotoxin were observed in the serum of mice injected intravenously with 12 ^I-labeled ricin- an i-L y-2.1 , 26 hours after injection whereas unconjugated MoAb could be readily detected, up to 90 hours after injection. Accordingly free MoAb could act as a potential inhibitor of the binding of residual immunotoxin to the target cells in vivo. Recently Bourrie et al (10) reported that yeast mannan could enhance the level of a ricin- A-chain immunotoxin in circulation by inhibition of liver uptake and thus increased the anticancer - C17 - (T-'Γ-) efficacy of immunotoxin in vivo. It is likely that such b locking agents can also be used with ricin-antibody conjugates. REFERENCES PART C 8. SMYTH l-IJ, PIETERSZ GA, CLASSON B, et al. Specific targeting of chlorambucil tumors with the use of monoclonal antibodies. JNCI 1985; 76:503-510. 9. FOLEY GE, LAZARUS H, FARBER S, et al. Continuous culture of human lymphoblasts from peripheral blood of a child with acute leukemia. Cancer 1965; 18:522-529. 10. BOURRIE 8, CASELLAS P, BLYTHMAN H, et al. Study of the plasma clearance of a n t i b o dy - r i c i n - A - c h a i n immunotoxins. Evidence for specific recognition sites on the A chain that mediate rapid clearance of the immunotoxin. Eur J. Biochem 1986; 155:1-10. 11. WEIL C. Tables for convenient calculation of median- effective dose (LD50) or ED50) and instruction in their use. Biometrics 1952; 8:249-254. 12. KNAlil 8, C00NEY J, EDINGT0N T, et al. Differences in experimental tumor localization of dual-labeled monoclonal antibody. J Nucl Mad 1986: 27:1293-1299. 13. F00STAD 0, KVALHEIN G. G0DAL A, et al. Phase I study of the plant protein ricin. Cancer Res 1984; 44:862-865. 14. ANDERSON M, MEISTER A. Dynamic state of glutathione in blood plasma. J Biαl Chem 1980; 255:9530-9533. - c /γ - Cht) Table I - Biodistribution of ^I-labeled r _ c in and modified ricin 30 minutes after intravenous injection into B6CF j mice. 1 Z Initial dose X Initial dose per per organ gram tissue Tissue Ricin Modified Ricin Modified Ricin Ricin Blood 9.0 21.5 4.3 10.3 Liver 35.7 8.1 23.1 4.9 Spleen 3.4 3.1 24.4 25.9 Kidney 2.9 4.7 7.2 10.2 Heart 0.7 0.8 4.5 4.6 Mice received an injection of 0.12 yg toxin (4.6-5.8x10° cpm/μg) Table C -2- Biological activity of native ricin, modified ricin, ricin- antibody and modified ricin-antibody conjugates. The results show the dose of ricin in ιg/kg body weight calculated to kill 50% of the mice. Treatment Toxicity to Mice LD50 i.p. i.v. Ricin 1 .7 ' 0.8 Ricin-MoAb 7.0 4.5 Modified Ricin - 0.4 Modified Ricin-MoAb - 8.3 - D2 - t+W 1 blood activity was calculated on the assumption that the 2 overall blood volume represent 7? of total body weight. Mice 3 were sacrificed, tumor removed, weighed and the radoactivity 4 measured and calculated as a percent of the initial injected 5 dose . • - • I2i2££ l£££Aϋ!££i ϋAiil £2__QJ£2£i££ 7 In tumor growth studies, mice bearing solid tumors (0.2 to 8 0.6 cm 2 in size) were treated by IT injection and the size 9 of the tumors measured daily along the perpendicular axes of 10 the tumors; the data were recorded as mean tumor size (the 11 product of two diameters - standard error of the mean). 12 Experimental groups of up to 40 mice al l of the same sex and 13 age were used in each experiment and mice were observed for 14 up to 200 days. "• s An v Ai £ £Aπ__A£2 ϋ£2£Z 16 A binding assay previously described in Part B was used to 17 determine the reactivity of ' 25 I - a n t i-L y - 2.1 to E3 tumor 18 cell lines r e-es t ab 1 ih sed in culture after IT treatment with 19 ricin- aπti-Ly-2.1. 20 Histolojai 21 Sections from the tumors, spleens, livers and kidneys were 22 fixed in 10? formalin and stained with haematoxylin and 23 eosin for examination under light microscopy. 24 RESULTS 1 - _ 25 £A£££££££ £__ I- l£^£A££ ' !!_£__.__. -.H E EAE f££JB l£E££ 26 B6CF1 mice bearing E3 (Ly-1~, 2 + ) thymoma grafts received an 27 IT injection of 25 I-labeled anti-Ly-2.1 , anti-Ly-1.1 , or 28 ricin and the clearance of iodinated material was determined 29 from the relative amount of radiolabel in the tumor and in 30 the blood. The clearance from tumors of two MoAb and ricin 31 in B6CF1 mice waβ examined (Figure D 1 )-, 3 groups of 30 mice 32 injected; 6 mice from each group were sacrificed at 33 different time intervals (1 ,8,21 ,29,51 houxs) and the 34 distribution of the injected radioactivity measured. A3 35 shown in Figure D1a, 45-53? of the initial injected MoAb was 36 retained by the tumor 1 hour after injection. The reactive 37 ( a n t i-L y- 2.1 + ) MoAb was retained by the tumor at a higher 38 concen ration for a longer time, and 53? of the initial - oi - (H> Part D This Part evaluated the use of whole ricin-an i od conjugates as therapeutic agents for cancer. Iilith the use of IT injection, therapeutic doses of whole ricin could be localized to tumors. The ability to successfully use whole ricin was due to the loss of the galactose binding site on coupling of ricin to antibody to produce conjugates with little nonspecific binding (described previously in Parts 8 and C ). MATERIALS AND METHODS Mice Athymic, BALB/c mice (nu/nu) were obtained from the Royal Dental Hospital (Melbourne) and were maintained in specific p a t hoge n f r e e c ond i t ion s. T h e i n b r ed s t r a in (C57BL/6xBALB/c)F1 (B6CF1) mice were maintained in the animal colony, Department of Pathology, The University of Melbourne. l£ £ ££ l The human T-cell leukaemia CEM (14) and the colon carcinoma HT-29 (15) were maintained in culture. CEM cells (2x10p 7 ) or HT-29 cells (5x10 6 ) were injected s u b c u a n e o u s 1 y into the abdominal wall of nude mice. The murine thymoma E3 (16) was maintained by serial passage αf ascites fluid in B6CF1 mice. For in vivo experiments, E3 cells (2- 3x10 ) were injected s ubcutaneous 1 y into B6CF1 mice. £££££££__ • A££ £__ -_A£A£__£π__A__£___- £££__£££-_££ Ricin (Sigma Chemical Company, St. Louis, M0, USA) was conjugated to MoAb by a disulf ide bridge using the h eterobif unctiona 1 crossliπkers SPDP an SAMSA (Part 8). £A£££££££ S.L •- ■ ~ IEEAEE " £_£j_.__2 £J2£l EAEAE A£i££__£2 ' AH_2 __£-_2££ Ricin (30 mug, 1 mg/ml) in PBS containing 100 M lactose and MoAb (30 mug, lug/ml) were labeled with 25 I using chloramine T (17) , to a specific activity of 2-3x10° cpm/ mug and 4.6x10 cp / mug respecti ely. Groups of 6 B6CF1 mica all bearing established E3 thymomas (0.6-0.8 cm in size) received IT injections of MoAb (2.0 mug) or ricin (0.5 mug); blood samples were taken at different times, and the - D3 - ( S '" ) injected dose u a 3 found in the tumor 8 hours after injection; 31? of the initial dose remained 21 hours after injection. By comparison the clearance of the nonreactive MoAb was faster and only 15? of the initial dose was found in the tumor 8 hours after Injection and only 6? of the initial dose remained 21 hours after injection. The decrease in tumor radioac i ity observed for the nonreactive MoAb coincided with a proportional increase in blood activity and 21 hours after injection 49? of the initial injected activity was found in the blood, by contrast the blood activity observed for the reactive MoAb was less than 4? at all ime intervals measured. til h 01 e ricin alone, by virtue of its galactose binding property was cleared more slowly from the tumor than the nonreactive MoAb but faster than the reactive MoAb. As shown in Figure D1b, 29? of the initial injected dose was retained by the tumor 1 hour after injection. The amount retained by the tumor decreased with time and only 10? of the initial dose remained in the tumor 21 hours after injection and only 4? of the initial dose remained 51 hours after injection. Blood activity increased with time and 19? of the initial injected dose was found in the blood 21 hours af ter injection. £-_.-_££-_-_-_££ l£££____£2__ £__ £._.-, ____£A__£ I_2_d_2£__£ ϋAJi-i £ £A£A__-_ £πiA- 2 " £2π__iϋ2£i£ To determine whether intact ricin-antibody conjugates could be used for IT treatment, palpable tumors of 0.3-0.4cm in size were treated. In this study groups of at least 10 mice received an IT Injection of one of the following treatments in 100 mul of PBS: (i) PBS; (ii) anti-Ly-2.1 ; (iii) ricin- aπti-Ly-2.1 ; (iv) ric in - a n t i -L y- 1.1. In a representa ive experiment mice bearing tumors received two injections directly into the tumor on days 7 and 9 with preparations containing 0.2 mug ricin and 0.9 mug antibody. As shown in Figure D2a, the ricin-anti-Ly-2.1 treated tumors regressed and within 48 hours al l tumors had virtually disappeared, while untreated E3 tumors grew to a large size (4.5-5.0 cm 2 ). The antitumor effect of the ricin-anti-Ly-2.1 - D4 - (SO conjugate was specific since there was no significant inhibition of tumor growth in the groups which received MoAb alone or r iciπ-an i-Ly-1.1 (Figure 02a), which did not bind E3 cells (the anti-Ly-1.1 conjugate treatment gave some inhibition of tumor growth but the tumors grew on relentlessly and the mice dies). Figure D2b shows the individual growth curves of the mice treated with ricin- anti-Ly-2.1 and complete regressions were observed in 4/10 mice which remained tumor free for greater than 200 days. In 6/10 mice the tumors regressed for 2-4 days and then grew again, usually at the original site. Iilheπ tracing the individual tumor growth curves of the mice treated with r ic i n - a n i- -1.1 , no complete regressions were obtained (Figure 02c). Autopsy of the animals in the ricin-anti-L'y- 2.1 treated group and histological examination of organs (liver, kidneys, spleen) did not reveal any tissue damage. Animals in the group treated with the nonreactive conjugate showed damage to the liver, spleen and kidneys, presumably as these conjugates were not retained by the tumor. __££E_!£A£2-_ £____ _iA£i2A22__ 9. l£££i£i_! ££!_! -.A Tumors Regression was characterized morphologically and histα 1 ogica 11 y in mice bearing E3 tumors which were treated IT with the ricin-antibody conjugates. Mice were examined 17 days fallowing treatment and a comparison of the size of a ric in-an ti-Ly-2.1 treated tumor to a tumor treated with antibody alone was made. The r i c I n - a n t i - L y - 2.1 treated tumors shrank to a dry black scar that eventually separated leaving a clean shallow ulcer at the injection site, whereas tumors treated with MoAb alone grew to 5.0cm 2 in size. Several (2/4) cured mice were sacrificied 30 days after treatment and were autopised to ensure that there was no tumor regrowth and showed no pathological evidence of tumor. Of 2/6 mice treated with r i c i n - a n t i - L y - 2.1 where the tumors recurred, one should note that the tumor re- appeared around the initial injection site. The treated and untreated tumors were analyzed histo 1 og ica 11 . Untreated E3 tumors were wel l vascularized and were - D5 - (SZ) populated with viable cells, and also showed numerous mitoses. By contrast, the ricin-anti-Ly-2.1 treated tumors contained large areas of necrotic tissue showing much cellular debris with only small patches of viabl cells. Adjacent normal connective tissue and vascularized fat tissue were damaged. Some blood vessels contained thrombi. Together these observations suggested destruction of the E3 tumor cells by r i c i n- a n t i -L y - 2.1 conjugate and some minor damage in the immediate vicinity. l£££__l_£2_. £j_ lM__.£££ ωith (I0 ~/C BH 3 ~ i£££.-_£__l 5A£A____2£iA_!£-___ EEH.__i__.22.__22 The specificity and potency of a "modified" (10 ~ /CNBH " treated), ricin-anti-Ly-2.1 conjugate was undertaken in a tumor growth study to evaluate the antitumor effects when injected directly into established tumors. Groups of 10 B6CF1 mice bearing established E3 tumor 0.6 cm 2 in size received an IT injection in 100 mul of PBS either: (i) PBS; (ii) anti-Ly-2.1 ; (iii) modified ricin; (iv) modified r icin-a nt i-L y- 2.1. In this study the mice received a single IT injection of 0.88 mug of modified ricin and 4.0 g MoAb 5 days after tumor i nn oco 1 a t i on . As shown in Figure D5, there was significant inhibition of tumor growth in mice treated with conjugate and al l of the tumors regressed within 2 days after treatment but then regrew In 7/10 mice 11 days after the initial treatment. These recurring tumors were established in tissue culture and a binding assay showed the tumor cells to have the same distribution of Ly- 2.1 antigen as the parent £3 cell line. The binding curve obtained when ^ 25 I-anti-Ly-2.1 was incubated with the cells clearly showed that there was no noticeable difference In the uptake of radioactivi y by the 2 cell lines. Thus the tumor cel ls that escaped being killed in the conjugate treated mice were not a mutant subclone deficient in Ly-2.1 antigen. Furthermore, the serum of the treated mice was a n a l y z e d f o r a n a n t i - r i c i n r e s p o n s e u s i n g a r a d i o immu n oa s s a , but no ricin antibodies could be detected In the serum (not shown). Therefore these recurring tumors could then be retreated with a course of - D6 - LSI) 1 modified ricin-aπt i -L y - 2.1. 2 As a control, E3 tumors were treated either with 3 antibody alone or with modified ricin. Antibody when 4 Injected directly into the tumor had no effect on the 5 development of the tumors (Figure 05), whereas mice treated 6 with modified ricin (>0.3 mug) displayed symptoms of 7 po isonin g (we ig h t l os s , h emo rrh a ge , r e sp i r a to ry 8 difficulties, loss of balance) fol lowed by death 2 days 9 later. Lower doses of modified ricin (<0.2 g) did not kil l 0 the mice, nor did they cause tumors to regress. Thus a dose 1 of modified ricin (not attached to antibody) which was safe 2 to mice and destructive to tumors could not be found. 3 ££__E££A££_- 2.L ΪLS. £EiA-__J!I!££ £ff£ £s gf U EAB £Ei_ U UodAlAE^ (IO 4 " /CNBH 3 " ) RAEAE 5 The antitumor effects of the two different anti-Ly-2.1 6 conjugates were compared on estab lished tumor. B6CF1 mice 7 bearing thymoma grafts 0.6 cm in diameter were treated with 8 a single IT Injection 5 days after tumor innoculation. Al l 9 treatments contained equivalent amount of ricin and 0 antibody which were 1.0 mug and 4.6 mug respectively. The 1 results obtained are summarized in Table 01. 2 Bot h anti-L y-2.1 conjugat e s wer e ab l e t o caus e 3 regression of solid tumors. However the modified ricin-anti- 4 Ly-2.1 conjugate was a less effective antitumor agent than 5 the native ricin conjugate: modif ied r i c in- a n t i- y - 2.1 6 treatment cured 13/23 mice, which remained tumor f ree for 7 greater than 200 days. So l id tumors recurred in 7/23 mice, 8 which died due to tumor and 3/23 mice died due to ricin 9 toxicity. By contrast 24/30 mice treated with the nati ve 0 ricin conjugate survived tumor free (>200 days). One of the 1 mice developed a subcutaneous tumor at the site of injection 2 whereas 6/30 mic e de v e l op e d a scite s tumor s i n th e 3 p eritoneium. The mean survival time of these mice was 4 extended to 40 days compared to the 20 days mean survival 5 t ime of the PBS treated contro ls (i.e. r i c i π - a n t i - y - 2.1 6 treatment extended the surviva l time of the PBS treated 7 contro ls ( i.e. r i c i n - a n t i - L y - 2.1 treatment extended the 8 survival time of tumor bearing mice). - D7 - (S+) The antitumor effect of the two aπti-Ly-2.1 conjugates wa3 specific since antibody alone, ricin alone, and modified ricin alone were all without protective effect, and mice either died due to ricin toxicity or due to tumor. The conjugate treated mice that showed no signs of tumor growth after 200 days were either given an iπtraperitonea 1 or subcutaneous injection of 3x10 s E3 cells. The mice all developed tumors and died over the same period (18-20 days) as an age matched group of control animals. The conjugate treated mice had therefore not developed detectable resistance to the tumor cells. l£22_s__£E_i ° ______£__ !____££ 2_E.__£2££f £2 AE _5_.___.__E J3A22 To study the potentia l use of immunotoxins for therapeutic treatment of human tumors an experimental model of human neoplasia was used where intact ricin-antibody conjugates were used for IT treatment of human tumor xenografts in nude mice. Three studies were performed. £__E2£A__2I2.__ 1 : Groups of 4 nude mice with palpable CEM tumors 0.2- cm in size received a single IT injection in 100 mul PBS of either: (i) PBS; (ii) anti-TFR; (iii) riciπ-anti- TFR. Mice were treated 7 days after tumor inoculation with one of the preparations contaiπg 1 mug ricin and 4.6 mug antibody. As shown in Figure D7 all CEM tumors treated with r icin-anti-TFR regressed within 2 dayβ following treatment and there was no relapse even after 120 days. Injection of anti-TFR alone had no effect on CEM tumor growth. £__E2£AE2E__ __ ! Nude mice bearing human colon carcinoma cell line (HT-29) xenografts were used. Three groups of 5 mice were treated as follows: (i) The first group received a reactive ricin-17.1 /2 conjugate; (ii) the second group a nonreactive ricin-aπti-H A conjugate, our HT-29 cells are nonreactive with our anti-HLA; (Iii) the third, the control group, received P8S alone. Each reagent was administered directly into established tumor 0.3 cm 2 in size, 10 days after tumor inoculation. The results clearly demonstrate complete tumor regression in the group that received the ricin-17.1 /2, and this was apparent after only 2 days (Figure D8). To determine whether regression of HT-29 - D8 - CS5) tumors was due to the specific binding of the immunotoxin through the antibody moiety, HT-29 tumors were also treated with an irrelevant nonreactive control immunotoxin, riciπ- anti-HLA which did not bind HT-29 cel ls. There was no significant inhibition of tumor growth (Figure D8). Thus the specificity of ricin-antibody conjugate IT therapy is proven. In a comparison of a ricin-17.1/2 treated tumor at 120 days after IT injection to an untreated mouse bearing a HT-29 tumor 40 days after tumor inoculation it was noted that only a fibrotic scar remained at the previous location of the ricin-17.1 /2 treated tumor indicated by the arrow, whereas the untreated tumor measured 1.4 cm in size. iE £ E£Ei J To determine whether intact ricin- antibody conjugates could be effective on large tumors, mice bearing HT-29 tumors 1.5-2.0 cm 2 In size (1.2-1.4 cm in diameter) were treated with one of two conjugates: (i) reactive ricin-17.1/2; (ii) nonreactive riciπ-anti-H A. Mice received an in ra u ora 1 injection containing 1 mug ricin In 100 mul PBS 40 days after tumor inoculation and this was repeated 3 days later, as indicated by the arrows in Figure 9. Al l three HT-29 tumors treated with ricin-17.1 /2 regressed, and there was no relapse. When ricin-anti-H A was administered HT-29 tumors did not regress. DISCUSSION Selected MoAb should be suitable vehicles for carrying toxins such as ricin to tumor cel ls because of their specificity (by motive of their monoc 1 ona 1 it y) and ability to be produced in virtual ly unlimited amounts. Part B demonstrated that such conjugates are indeed powerful reagents in in vitro systems but obviously, the most attractive use of the conjugates are as a therapeutic agent for cancer in patients. However the in vivo application of these conjugates was not as dramatic as expected, .and a number of limitations were noted such as toxicity towards nontarget tissue, instability, and rapid plasma clearance. The localization of the conjugate to the tumor is an obvious critical step to achieve in any in vivo therapy and the relatively short circulation life of whol e ricin- - D9 - csη antibody conjugate demonstrated in Part C and also reported by a number of other laboratories (18-20) may explain the reasons for the failure of In ra venous l y injected riciπ- antibody conjugates to show greater antitumor acti vity to solid tumors. To improve the action of these conjugates on tumors the amount of conjugate l oca l ized to a tumor was increased by the direct injection of the conjugate into estab lished tumors. The studies in this part showed that a therapeutic dose of whole ricin-antibody conjugate could be localized to solid tumors and the cytotoxic efficacy of the conjugate could be dramatically improved when the treatment was administered directly into the tumor. There were three unique f eatures of this study that wil l be separately discussed. The first is the mode of conjugate administration where conjugates were injected directly into and around solid tumors In mice. Secondly studies showed that MoAb could be specifically localized to tumor fol lowing regional injection; and finally therapeutic experiments were carried out which demonstrated that regional l y injected ricin- antibody conjugate was a potent antitumor agent which was effective in causing the regression of established tumors. The direct injection of whole ricin-an ibody conjugate into tumors gave some remarkable results. Tumors disappeared r apidly a lthough a proportion reappeared (Figures 02 and D5). Modification of the Injection protocol , which included injecting around the edge of the tumor and using multiple treatment s l ed t o a high p rop ortion of tumors bei ng completely eradicated (Table 01 ). The most important finding in these studies was that estab lished E3 thymomas (L ' y-1 ~, 2 + ) treated with a reactive r i c i π - a n t i - y - 2.1 conjugate regressed, whil e similar sized tumors treated with a nonreactive r ic in - a n t i-L y - 1.1 conjugate did not and thus selectivity was observed without lactose being present. Similar results were observed in the nude mouse model. Estab lished CEM (Figure 07) and HT-29 (Figure 08) tumors 0.2-0.4 cm 2 in size treated with r i c i n - a π i - T FR and ricin- 17.1 /2 respectively regressed. HT-29 tumors 1.5-2.0 c in 3ize were given two injections of ricin-17.1 /2 conjugate - D i o - C S V three days apart, and treatment was administered in this manner to saturate the tumor with immunotoxin and prevent recurrence due to tumor cells which may have escaped the first treatment. Under these conditions, HT-29 tumors treated with ricin-17.1/2 conjugate regressed wherease tumor treated with a nonreactive conjugate did not (Figure 09). In all studies, the regression of tumors was marked by a rapid decrease in- the mass of tumor which remained a flat, fibrotic scar. Light microscopy showed that this scar resulted from organization of the n ecrotic t issue. Furthermore examination of spleen and liver did not reveal any evidence of damage. As a control, ricin injected alone in doses equivalent to conjugate doses were lethal to mice two days after treatment (Figure D5). Death was preceded by weight loss, sk i n hemo rrage s , re sp i r ato r y a n d n er v o u s sy s t em disfunctions. The spleen and liver of these mice were damaged , findings in agreement with earlier light microscopic descriptions of damage caused due to ricin poisoning. A therapeutic effective dose of whole ricin alone could not be found and a dose of 0.20 mug or lower of ricin did not kil l tumor bearing mice but also did not cause tumor regression. Thus in contrast to reactive ricin-antibod conjugates, nonreactive conjugate and ricin alone were not effective antitumor reagents. Thi3 study has sho.n that the antibody not only served to target ricin to the tumor but antibody holds the ricin in the tumor and very little escapes. For example, 53? of the initial injected dose of a reactive MoAb could be localized to established tumor 8 hours after injection (Figure D1a). Thus the high affinity of the MoAb contributed to the localization of the reactive ricin-antibody conjugate to the tumour. By contrast conjugates using antibody which did not bind or ricin alone which has a lower binding coefficient are able to leak out of the tumor (Figure D1 ) and cause some nonspecific toxicity to the eticu1 oendothe 1 ia 1 system. In conclusion, this form of therapy may have some practica l v alue for treating superficia l tumors and - 01 1 - ( ζ$) recurrences of tumors in siteβ which are difficult to remove surgica l ly yet are amenable to injection. (Surgica l col leagues have suggested fixed regional 1 ymp h a d e n op a h causing pressure symptoms, hepatic metastases causing pain or obstructive jaundice and locally recurrent breast cancer or melanoma nodules. It should be noted that the clinical management of locally recurrent tumors is a problem which is currently treated by radiation where damage to surrounding normal tissue occurs conco i an 1 y. It is clearly important to fully explore the development of whole ricin-antibody conjugate for intratumor treatment which should be effective against only the tumor tissue. - D12 - (.$ ) REFERENCES PART D 1 . Foley, G.E., Lazarus, H., Farber, S. et al. Continous culture of human lymphocytes from peripheral blood of a child with acute leukaemia. Cancer 18:522-529 (1965) . 1 5. Fogh, J. and Trempe, G. New human tumor cel l l ines. In: Fogh, J., ed. Human tumour cel l s in vitro. New York : Pl enum Press, pp 115-120 (1975). 16. Smyth, M.J., Pietersz, G.A., Classon, B.J. et al. Specif ic targeting of ch lora bucil to tumor with the use of monoclona l antibodies. Journa l Nat l. Cancer Inst. 76:503-510 (1986). 17. Greenwood, F.C. , Hunter, lil.M. and Glover, J.S. The preparation of 1311-labeled human growth hormone of high specific radioactivity. Biochem. J. 89:114-123 (1963) . 18. B lythman, H.E., Casel las, P., Gros, 0. et a l. Immunotoxins: hybrid molecules of monocl ona l antibodies and a toxin subunit specifical ly kil l tumor cel l s. Nature 20:145-146 (1981 ). 19. Trowbridge, I.S. and Domingo, O.L'. Anti- transf errin receptor monoclonal antibody and toxin antibody conjugates a ffect growth of human tumor cel l s. Nature 294:171 -173 (1981 ). 20. Bourke, 8.J.P., Case l las, P., B lythman, H.E. et al. Study of the plasma clearance of an t ibody- r icin- A-chain immunotoxins. Eur. J. Biochem. 155:1 -10 (1986). WO 87/03286 - o . > ( t o) T/AU86/00370 TABLE P / WI TH MπΛb NATIVE KTCP- - MOM HIED RlCIM OR CSWUEftlFS. IR_ATMEMΓ OF E3 TUCUHS % Cure Treati-enL Mice No. Tυmwr oxici.y Solid Asr.ites Tλirour T 0 0 PHS: 10 10 (1007.) 10 6 80 anci-2.1- . (1:1) 30 1 (37.) ant l -2.1-R (1:2 ) 20 10 (50.) 10 50 (30_. anti_-2.1-R (Na.O. ) 23 7 7 3 (13%) 56 - 10 0 Ricin 10 o l_Ab 10 10 10 0 Native RJcin 10 diameter) wr p given injections intratuπorly with preparations Mice bearing E3 tumors (0.6 an in containing lug of ricin and 4.6 ug of anti-Lv-2.1 in 100 ul oi PBS

Claims: - 1 - ( fe -) 1, A conjugate of ricin being whole ricin or a derivative of ricin, which derivative per se being inhibitory of protein synthesis in cel l free systems, with an antibody which is preferen ial l absorbed by a tumour cel l as compared to a noπ tumour cel l and wherein the galactose binding site of said ricin is blocked and pharmacologically acceptable salts thereof. 2. A conjugate as c laimed in claim 1 , wherein the conjugation is via a disulphide linkage. 3. A compound of formula Ab-Y -X.- -NHR- j wherein Ab and the Y group attached thereto represent an antibody radical and wherein Y is S or NH, R^ and the amino group to which it is attached represents a ricin radical being a whole ricin radical or a radical of a derivative of ricin which derivative per se being inhibitory of protein synthesis in cell free systems and X^ , or Y-X- j (when Y is S), represent a conjuating chain including a disulphide linkage and wherein the galactose binding site of said ricin radical is blocked and pharmacologicall acceptable salts thereof. 4. A compound of formula I set out in the accompanying drawings wherein Ab and the amino or sulphur to which it is attached represents an antibody radical, R . and the amino group to which it is attached represents a ricin radical being a whole ricin radical or a radical of a derivative of ricin which derivative per se being inhibitory of protein synthesis in cell free systems, Z ^ and Z 2 which may be the same or different are H, alkyl, aryl, carboxy, halide, cyano, ester, carboxamide, sulphonate, hydrαxy or amino, n.. and n 2 which may be the same or different are 1-10, X 2 ar| d X 3 which may be the same or different are NH, 0 or S and Z is 0 or 1 and wherein the galactose binding site of said ricin radical is blocked and pharmacologicall acceptable salts thereof. 5. A compound as claimed in claim 4, and of formula II as set out in the accompanying drawing s wherein Ab-S- represents an antibody radical, R .. _ Nh" - represents a ricin radical being a whole ricin radical or a radical of a derivative of ricin which derivative per se being inhibitory - 2 - ((* _) of protein synthesis in cell free systems, and -R 2 -NH and NH-R- j -, which may be the same or different, are of formula IV as set out in the accompanying drawings wherein X^ is 0, S or NH, n is from 1 to 10 and Z 3 is hydrogen alkyl, aryl, carboxyl, halide, cyano, ester, carboxamide, sulphonate, hydroxy or amiπo. 5. A compound as claimed in claim 4, and of formula III as set out in the accompanying drawings wherein Ab-N represents an antibody radical, R.--NH- represents a ricin radical being a whole ricin radical or a radical of a derivative of ricin which derivative per se being inhibitory of protein synthesis in cell free systems, and -R 2 -NH and NH-R.,-, which may be the same or different, are of formula IV as set out In the accompanying drawings wherein X^ is 0, S or NH, π is from 1 to 10 and Z 3 is hydrogen alkyl, aryl, carboxyl, halide, cyano, ester, carboxamide, sulphonate, hydroxy or amino . 7. A compound as claimed in claim 4 and of formula V set out in the accompanying drawings wherein Ab-NH- represents an antibody radical and R.--NH represents a ricin radical being a whole ricin radical or a radical of a derivative of ricin which derivative per se being inhibitory of protein synthesis in cell free systems. 8. A compound as claimed in claim 4 and of formula VI set out in the accompanying drawings wherein Ab-NH- represents an antibody radical and R , | - NH represents a ricin radical being a whole ricin radical or a radical of a derivative of ricin which derivative per se being inhibitory of protein synthesis in cell free systems. 9. A compound as claimed in any one of claims 3-8 wherein said ricin radical is a whole ricin radical. 10. A compound as claimed in any one of claims 3-8 wherein said antibody radical is a radical of an antibody showing specificity for a selected one of breast, brain, melanoma, lung, pancreas and colon tumors. 11. A pharmaceu ical composition comprising a compound in accordance with any preceding cl aim together with a pharmaceutically acceptable diluent. - 3 - [ ( o3) 12. Compounds of formula VII, VIII or IX set out in the accompanying drawings wherein P is of formula VII a, VII b or VII c set 'out in the accompanying drawings and R^ t Ab, X , X3, Z 1 and Z , and n^ and n have the meaning given in claim 4. 13. A method of making a compound in accordance with any preceding claim comprising reacting whole ricin or a derivative of ricin which derivative per se is inhibitory of protein synthesis in cel l free systems to obtain a ricin containing a -S-S- group, reacting an antibody to obtain an antibody containing a -S-S- group, reducing one of the conjugates to form a -SH group and reacting the reduced conjugate with the other of the conjugates to obtain the compound desired. 14. A method of making a compound in accordance with any one of claims 1 -12 comprising utilizing a sulphydryl introducing compound to produce a side chain on an antibody or whole ricin or a derivative of ricin which derivative per se is inhibitory of protein synthesis in cell free systems containing a -S- linkage, reducing that linkage and then reacting to form an antibody ricin conjugate containing a -S-S- linkage. 15. A compound in accordance with any one of claims 1-12 or a method of making same or a pha aceutical composition containing same substan ially as hereinbefore described with reference to any one of the tests, experiments or examples herein . 15. The articles, things, parts, elements, steps, features, methods, processes, compounds and compositions referred to or indicated in the specification and/or claims of the application individually or collectively, and any and all combinations of any two or more of such.

Inventors: KANELLOS JERRY, MCKENZIE IAN FARQUHAR CAMPBELL, PIETERSZ GEOFFREY ALLAN, KANELLOS, JERRY, MCKENZIE, IAN, FARQUHAR, CAMPBELL, PIETERSZ, GEOFFREY, ALLAN
Applicants: UNIV MELBOURNE, THE UNIVERSITY OF MELBOURNE
Application year: 1986
Main classification: C07K15
IPC codes: A61K47