Pharmacokinetics of Tumor-reactive Immunotoxins in Tumor-bearing Mice: Effect of Antibody Valency and Deglycosylation of the Ricin A Chain on Clearance and

Tumor Localization
R. Jerrold Fulton, Thomas F. Tucker, Ellen S. Vitetta, et al. Cancer Res 1988;48:2618-2625.

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[CANCER RESEARCH 48, 2618-2625, May 1, I988|

Pharmacokinetics

of Tumor-reactive

Immunotoxins in Tumor-bearing

Mice: Effect

of Antibody Valency and Deglycosylation of the Ricin A Chain on Clearance and Tumor Localization1
R. Jerrold Fulton, Thomas F. Tucker, Ellen S. Vitetta,2 and Jonathan W. Uhr
Department of Microbiology, Southwestern Medical School, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235

These studies have established the basic pharmacokinetic prop erties of ITs. IT-As are cleared very rapidly from the circulation; The blood clearance and tissue distribution of immunotoxins composed within l h after injection only 10-30% of the material remains of either intact or Fab' fragments of tumor-reactive, monoclonal rat antiin blood (11-13) and the major organ for uptake of IT-As is murine immunoglobulin D (anti-<5)or nonreactive, normal rat inimiinothe liver (13). This rapid binding by cells in the liver is mediated globulin G and ricin A chain (native or chemically deglycosylated) were largely through binding of the A chain to mannose-specific determined in B( I,, tumor-bearing mice. In the presence of accessible target cells, neither the valency of the antibody nor deglycosylation of receptors on nonparenchymal cells (16) since coinjection of the A chain affect the initial rate of clearance (a phase) of immunotoxins mannan with IT-As (12) or partial deglycosylation of the A from the blood; however, both factors affect the levels of tumor-specific chain (13) prevents the majority of liver uptake. The remaining and nonspecific localization of the immunotoxins. Thus, the use of 10-30% of the IT-A is cleared more slowly with a half-time immunotoxins with deglycosylated A chain greatly reduced the levels of (i./,)of 5-10 h ( 11-13). Depending on the nature of the disulfidenonspecific uptake by the liver and concomitantly increased tumor- containing linker between the antibody and the A chain, the specific localization. Immunotoxins prepared with Fab' fragments of IT-A may gradually break down in the circulation (13, 15, 17); anti-A antibody and deglycosylated A chain were twice as effective at however, there is disagreement on this issue (12, 14, 18). localizing to splenic tumor than immunotoxins prepared with intact With few exceptions, the studies discussed above have been immunoglobulin G and deglycosylated A chain. Approximately 90% of performed in normal animals using IT-As which do not react tumor-specific localization of anti-A immunotoxins occurred within l h after injection and less than 5% of the immunotoxin remained in the immunologically with tissues in the animal. Since a major circulation 4 h after injection. In addition, the antigen-binding capacity objective of research on ITs is to use them to treat human of the remaining circulating immunotoxins decreased in a linear manner tumors i/i vivo, it is important to characterize the fate of tumorover the first 10 h to approximately 20% of the initial binding activity. reactive IT-As in tumor-bearing animals and, in particular, to Thus, by 10 h after injection, only 2-3% of injected immunotoxin re determine the ability of IT-As to localize specifically in tumor mained in the circulation and this material expressed little antigen tissue after systemic administration. In the present study, we reactivity. Analysis of the in vivo stability of circulating '"'I-labeled have examined the pharmacokinetics and tumor-specific local immunotoxins in tumor-bearing mice demonstrated that Fab' immu ization of tumor-reactive IT-As in mice bearing the BCLi notoxins are more stable than IgG immunotoxins prepared with N- tumor. The BCLi tumor is a surface IgD+ B-cell tumor that ABSTRACT
succinimidyl-3-(2-pyridylthio)propionate. In BCLi-bearing mice, signifi cant splitting of the unli-5 immunotoxins did not occur until tumor localization was virtually complete.

INTRODUCTION Conjugates of antibodies or antibody fragments with protein toxins or toxin subunits (ITs3) have been prepared for the purpose of killing subsets of normal and neoplastia cells (re viewed in Refs. 1 and 2). Many studies have utilized the toxic subunit (A chain) of the plant toxin ricin to prepare ITs. IT-As can be highly effective cytotoxic agents for target cells in vitro (1-6) and in some cases in vivo (7-10). It is of obvious importance to determine the biodistribution of IT-As prior to their use in tumor-bearing patients. There are numerous reports of the /'// vivo clearance and tissue localization of IT-As (11-13) and of ITs containing gelonin, saporin, and pokeweed anti-viral protein (all of which are A chain-like ribosome-inactivating proteins) in rodents and primates (14, 15).
Received 9/28/87; revised 1/20/88; accepted 1/29/88. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work is supported by NIH Grants CA-28149 and CA41081, a grant from the Welch Foundation (1-947), and a grant from the Meadows Foundation. 2 To whom requests for reprints should be addressed, at Department of Microbiology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry HiesBlvd., Dallas, TX 75235. 1 The abbreviations used are: IT, immunotoxin; A, native ricin A chain; dgA, chemically deglycosylated ricin A chain; IT-A, IT containing native ricin A chain; IT-dgA, IT containing deglycosylated ricin A chain; SPDP, JV-succinimidyl 3-(2pyridyldithio)propionate; NRtlg, normal rat IgG; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

grows primarily in the spleen and progresses to severe leukemia later in the disease (19). The results of this study demonstrate that IgD-reactive (anti5) IT-As are removed from the blood of BCLi mice extremely rapidly and that the nature of the antibody (Fab' or IgG] or the A chain (native or deglycosylated) has little effect on the rate of clearance when the IT-A is targeted to accessible tumor cells. The majority of both tumor-specific and nonspecific tissue binding occurred within the first hour after injection and ac counts for the removal of 80-95% of IT-As from the circulation. The rate at which the IT-As break down into free antibody in the circulation appears to be too slow to affect the majority of tumor-specific localization. The highest levels of tumor-specific localization were observed using specific Fab'-ITs prepared with dgA chain (Fab'-dgA).

MATERIALS

AND METHODS

Mice. Female BALB/c mice (6-8 weeks old) were purchased from Cumberland Laboratories. Mice bearing the BCLi leukemia were used 3-4 weeks after i.p. injection of 1 x IO7BCLi spleen cells. The average tumor burden in these mice was 5.0 x I(I*Id* spleen cells and less than 5x10* Id+ peripheral blood lymphocytes/ml as determined by indirect immunofluorescence and analsysis on the fluorescence-activated cell sorter (data not shown). Antibodies. JA 12.5 hybridoma cells secreting rat IgG2b anti-mouse 6 antibody were the kind gift of Dr. Joseph M. Davie, St. Louis, MO. Endotoxin-free, purified JA 12.5 antibody was produced by Damon Biotech, Inc. NRtlg was purified from normal rat -y-globulins (Sigma Chemical Co., St. Louis, MO) by ion exchange on carboxymethyl 2618

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PHARMACOKINETICS

OF IMMUNOTOXINS

IN BCL.-BEARING MICE

cellulose followed by gel filtration on Sephacryl S-200 (Pharmacia, Piscataway, NJ) as described previously (20). The IgG fraction con tained approximately equal amounts of rat IgG2a and IgG2b as deter mined by Ouchterlony analysis and by the staining intensity of the two distinct heavy chain bands following SDS-PAGE of samples in the presence of 2-mercaptoethanol (data not shown), l-'(ah'), and Fab' fragments of JA 12.5 and rat IgG were prepared by pepsin digestion and reduction as described previously (20). Antibodies and their frag ments were iodinated by the lactoperoxidase method to a specific activity of approximately 1 x IO6cpm/Vg (21). Ricin A Chain. Both native (A) and deglycosylated (dgA) ricin A chains were purchased from Inland Laboratories, Austin, TX, and were prepared and characterized as described previously (22, 23). For prep aration of dgA, intact ricin is treated with a mixture of sodium metaperiodate and sodium cyanoborohydride for 4 h at pH 3.5 as described (23). This procedure results in the oxidative cleavage of carbohydrate ring structures in both the A and B chains followed by reduction of the resulting aldehyde groups to stable primary alcohols (23). Prior to purification of dgA from deglycosylated ricin, the effectiveness of the deglycosylation procedure was determined by demonstrating that >90% of the treated ricin (as compared to less than 5% of native ricin) failed to bind to Sepharose-concanavalin A (S mg/ml Sepharose) in the presence of 0.1 M galactose. Sepharose-concanavalin A was prepared in the presence of mannose at a ratio of 5 mgconcanavalin A/ml packed Sepharose. ITs Prepared with I25I-labeled A Chain. IT-As containing '"I-labeled ricin A chain and unlabeled IgG or Fab' fragments were used for determination of blood clearance and tissue localization. Before iodination, the cysteine residues of the A chain were blocked with Ellman's reagent (24), as described previously for substitution of Fab' fragments (20). Ellman's substituted A chain was iodinated with Na'"'I by the lactoperoxidase method (21) to a specific activity of 2 x 10" cpni///g. '"I-labeled A chain was reduced and desalted and a 5-fold molar excess of A chain was conjugated with PDP-substituted IgG or Ellman'ssubstituted Fab' fragments as described previously (20). The IT-As were purified by gel filtration on a column of Sephacryl S-200 (2.5 x 90 cm, equilibrated in PBS). The peak containing IT-A was pooled and unconjugated IgG or Fab' fragments were removed by chromatography on Blue Sepharose (Pharmacia) (25). IT-'25I-As were analyzed by SDS-PAGE under reducing and nonreducing conditions (26). The intensity of bands detected by staining with silver nitrate and by autoradiography was quantified on a Bio-Rad model 620 densitometer (Fig. 1; Table 1). Blood Clearance and Organ Distribution. Sodium iodide (10 /jg/ml) was added to the drinking water of mice 24 h prior to injection. Mice were lightly anesthetized with ether prior to i.m. injection of 100 mg ketamine and 7.5 mg promazine/kg body weight. Approximately 5 x 10' cpm (5-10 ng) of '"I-labeled A chain, IT-'"I-A, 125I-IgGor '"IFab' fragments in 0.1 ml PBS were injected i.v. (into the retroorbital

sinus) and '"I levels were determined in blood and major organs at 10 min, 30 min, l h, 4 h, and 24 h. The blood clearance and tissue localization of each protein were determined in 2 mice at each time point (Table 2). Heparinized blood samples (75 ^1) were collected at the indicated times and plasma was obtained by centrifugation. The total radioactivity remaining in blood was determined by counting aliquots in a gamma spectrometer and assuming a total blood volume of 7% of body weight (27). Acid-precipitable radioactivity was determined by precipitation of plasma aliquots with 10% trichloroacetic acid. The percentage of the injected radioactivity remaining in the circulation is presented as the percentage of acid-precipitable radioactivity injected (percentage of injected dose). The rates (rw values) for both the a and phases of clearance were determined using a computerized least squares regres sion analysis. Based on the major inflection point in the clearance curves (Fig. 2), the a phase was designated as 0-1 h and the phase as 1-24 h. The organ distribution of iodinated proteins was determined follow ing perfusion with PBS. Anesthetized mice were immobilized on a surgical surface so that the perfusate could be collected. After the thoracic cavity and pericardia! sac was opened, a catheter (connected to a reservoir of PBS) was inserted near the apex of the left ventricle. The right atrium was cut and a flow rate of approximately 20 ml/min was maintained until the perfusate was clear and the lungs and liver were very pale (approximately 5 min). Organs were removed, weighed, and counted in a gamma spectrometer. The luminal contents of diges tive organs were removed and processed separately. Radioactivity in skeletal muscle was calculated from samples assuming total muscle weight of 40% of body weight (27). In Vivo Stability of IT-As. '"I-labeled IT-As were prepared and the in vivo stability of Fab' and IgG-IT-As was determined by following the appearance of free '"I-Fab' or '"I-IgG in the circulation after injecting the iodinated IT-As. Fab'-anti-a-dgA and IgG-anti-a-dgA were iodinated with Na'"I by the lodogen method (28) to a specific activity of 1 x IO7 cpm/ng. The proportion of radioactivity present in the antibody portion of the iodinated IT-As was determined by scanning the autoradiographs following SDS-PAGE of reduced samples. For 125I-IgG-A,approximately 83% of the radioactivity was present in the bands corresponding to heavy and light chains; for '2!I-Fab'-A, approx imately 65% of the radioactivity was present in the bands corresponding to the Fd heavy chain fragment and light chain (data not shown). Similar analysis of unreduced samples showed that less than 5% of the radioactivity was present in the form of unconjugated IgG or Fab' (data not shown). After i.v. injection of approximately 5 x IO8cpm, plasma samples were collected at 15 min and 2, 4, 8, 12, 24, and 48 h. Plasma samples were analyzed by SDS-PAGE under nonreducing conditions followed by autoradiography of the gels. The bands corresponding to IT-A and antibody were quantitated by densitometric scanning and integration using a Bio-Rad model 620 video densitometer. In order to compare the IT-As and free antibody on a molar basis of antibody molecules, the values for IT-A were corrected for the fraction of radioactivity which was present in the antibody portion of the IT-A before injection. Based on the values obtained from SDS-PAGE (under reducing conditions) as discussed above, 83% of radioactivity in the IgG-A and 65% of radioactivity in the Fab'-A were present in the antibody portions. It was also necessary to correct for the longer halflives of free IgG and Fab', as compared to their respective IT-As. Both the IgG and Fab' accumulate over time, compared to the IT-As, thus increasing the apparent rate of dissociation of antibody and A chain. During the phase of clearance [2-24 h (Table 2)], the anti-5 IgG-dgA was cleared from the blood 2.1 times faster than anti--IgG [t* = 5.8 and 12.2 h, respectively]. Likewise, the anti-5 Fab'-dgA was cleared 1.6 times faster than Fab' (r* = 4.2 and 7.0 h, respectively) (Table 2). Thus, the molar ratios depicted in Fig. 6 were calculated as follows for each time point. IgG-A [IgG-A] x 0.83 IgG [IgG] - 2.1 Fab'-A _ [Fab'-A] x 0.65 Fab' [Fab'] - 1.6 (A) (B)

B
Mr
150* Mr

92* 68*
45*

92* 68* 45* 28* 22*

Fig. 1. SDS-PAGE of IT-'"I-As. ITs prepared with iodinated A chain and unlabeled IgG or Fab' fragments were analyzed on 7.5% and 10% polyacrylamide gels under nonreducing and reducing conditions, respectively, and stained with silver nitrate. A, nonreduced; B, reduced. Lane 1, Fab'-anti-6-A; Lane 2, IgG-antio-A. Ordinate, molecular weight in thousands.

2619

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PHARMACOKINETICS Table

OF IMMUNOTOXINS ofSDS-PAGE

IN BCL.-BEARING MICE on IT-'"/-/<

1 Densitometry

absorbanceSilver AutoradiographyIT Composition 82,000Fab' Fab'-A 52,000-60,000Fd 25,000-28,000A + L 30,000-32,000IgG-A chain 180,000-210.000IgG IgG-A 1-2 150,000H 25,000A + L 30,000-32,000 chain not detected; L, light chain; H, heavy chain.Table , M,Fab'-A stain Nonreduced81 7015 2258<2 <592 91271 50,000 and <1 <3A100

% of

Reduced " 40 28

2 Blood clearance of antibodies and IT-As in BCL,-bearing mice"a phase% phase circulationAntibody hAnti--* activity 2Anti--A 0.2Anti--dgA 0.4Rat 10Rat IgG 0.5Rat IgG-A 3Fab'-anti-IgG-dgA 2Fab'-anti-4-A 0.1Fab'-anti--dgA 0.4Fab'-rat 2Fab'-rat IgG 0.1Fab'-rat IgG-A 0.3A IgG-dgA in/. , (min) 13 11 12 47 34 51 14 12 14 31 23 37 remaining in the circulation at 1 h 8 5 6 34 25 38 10 11 10 22 12 25 /. (h) 12 5 6 22 4 6 7 4 4 6 4 4 .; % remaining the at 24 r\ r \li*.. IL1015Q. 10^~"~ .__ '* '
"tX\\^-~vv

100

*\
^\\\\\\ \
OQ 0.1UJHO

W\\\\V\i

0.1\:4"

UJ->

10Cz

O'l.

24

"'

24D:===8V,YV-*N\\\i

i4" ,,

0.3dgA chain 6 8 6 0.4" chain 9 16 5 usingthe Values were calculated by nonlinear least squares regression analysis ofindividuals blood samples from two mice for each protein. The variation mean of point.'JA12.5.Antigen-binding of the mean value for each time was less than 15%

rUJoceD_1010.1.01kU**~~~~^^iA 100r 100

__^ ^Q~~

" v^A

"v

of125I-Fab'-anti-6-dgA Assay. The antigen-binding (anti-IgD) activity vitroassay and '2'I-IgG-anti-5-dgA was assessed in an in normalBALB/c using spleen cells (positive) or thymocytes (negative) from byMason mice. The assay was performed essentially as described and Williams (29). Serial 2-fold dilutions of IT-'"I-A prepara plasmasamples tions (labeled in both the antibody and dgA chain portions) or ofthe taken at IS min and 2, 4, 8, 12, 24, and 48 h after injection of0.2 IT-125I-Aswere incubated with 1.0 x 10' cells in a total volume Afterincubation,containing 0.02% sodium azide for 90 min at 4C. ml of PBS with0.2 the cells were pelleted by centrifugation and washed for'"'I PBS containing azide, and the cell pellets were counted ml cold alwaysless a gamma spectrometer. Binding to thymocytes was using boundto 0.5% of input cpm and was subtracted from the cpm than splenocytes to obtain values for specific binding. The percentage of control binding in plasma samples was calculated on the basis of inputacid-precipitable beforeinjection cpm compared to the IT-I25I-A preparations asNonreduced % of plasma cpm bound x 100 % of IT-A preparation bound All assays were performed in triplicate using separate plasma samples from two mice for each 125I-IT-A.

\\\\Y\
0.11i

' ' 24HOURSFig.014~A~KJ:i1 1HOURSReduced9092B^3=4,^"""" 4" // 24 ' '

i4" ,,

ofradiolabeled 2. Plasma levels of antibodies and IT-As. Mice were given injections heparinizedtubes antibodies or IT-As and blood samples were collected in at 10 and 30 min and 1, 4, and 24 h later was obtained by centrifugation and acid-precipitable radioactivity was determined as described in asacid-precipitable Methods." "Materials and Results are presented the percentage of injectedmean radioactivity. Values represent thePlasma of duplicate bloodsamples Fab'-rat from two mice per protein (n = 4). A, Fab'-anti-o; B, IgG-anti-i; C, IgG; D, rat IgG. Unconjugated Fab' or IgG ().IT-A (),IT-dgA (A).

with the monoclonal antibody JA 12.5 (rat IgG2b/i anti-murine a chain), 80% of the total protein and 75% of the total radio activity were present in the band corresponding to Fab'-A (M, 82,000). Approximately 15% of protein and 20% of radioactiv ity in the Fab'-125I-A were present in multiple bands ranging in

RESULTS Characterization of lodinated IT-A. IT-As containing 125I-A chain were analyzed by SDS-PAGE followed by silver staining (Fig. 1) and autoradiography (data not shown); bands were quantified by densitometry (Table 1). For Fab'-'"I-A prepared

molecular weight from 52,000 to 62,000. This material repre sents free Fab', Fab'-A, in which the disulfide bond between the heavy and light chains has been reduced, and A chain dimers. For IgG-'25I-A, 90% of protein and 91% of radioactivity was present as IgG-A-containing 1 (80%) or 2 (10%) A chains

2620

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PHARMACOKINETICS OF IMMUNOTOXINS [N BCL,-BEARING MICE

For both IT-125I-As, as their mean content of radioactivity in the first 4 h, is under reducing conditions, radioactivity was detected only in presented in Table 3. The contribution of blood-borne radio the bands corresponding to ricin A chain (M, 30,000 and activity to tissue localization was eliminated by extensive car 32,000). When gels of reduced samples were stained with silver, diac perfusion prior to dissection of the tissues. Only four A chain represented 40% of the protein in the Fab'-A and 28% organs (spleen, liver, kidneys, and lungs) excluding total skeletal of the protein in the IgG-A (Table 1). The immunoreactivity of muscle and excretory sites contained greater than 2% of the all of the anti-5 antibodies and ITs was greater than 80% of injected radioactivity at any time point with all of the proteins acid-precipitable radioactivity, with the exception of unconju- examined. In contrast to the other organs which contained 60gated Fab'-JA12.5 which was 68% (data not shown). 90% acid-precipitable radioactivity, urine and stomach/intes Kinetics of Clearance of IT-As from Blood. The clearance of tinal contents contained greater than 90% acid-soluble radio IT-125I-A, IT-'25I-dgA or 125I-labeled antibody or Fab' frag activity indicating that these are pathways for excretion of ments from the blood of BCL, mice was examined after i.v. degraded A chain and/or free iodide. Since salivary and gastric injection of approximately 5 ^.g protein containing 2-3 x IO6 secretions represent the major body sites other than thyroid for cpm. In preliminary experiments, it was found that doses of the concentration of free iodide (30), the levels of acid-soluble Fab'-JA12.5-A containing 5 to 200 igf total protein did not radioactivity present in the gastrointestinal contents give some o indication of the rate of dehalogenation of the iodinated pro appreciably alter either blood clearance or tissue distribution (data not shown). In all cases, acid-precipitable radioactivity teins. These levels never exceeded 10% of the injected dose was used to calculate clearance rates. Fig. 2 shows the blood during the 4-h period. As shown in Fig. 3, the tumor localization of 125I-Fab' and clearance in BCLi tumor-bearing mice of Fab'-A (Fig. 2, A and 125I-IgG-anti-5 antibodies was very similar in the spleen which C), and IgG-A (Fig. 2, B and D) prepared with either anti-5 antibody (Fig. 2, A and B) or NRtlg (Fig. 2, C and D). Table 2 contains the majority of tumor; however, the levels of tumorsummarizes the tv, values of the phase (0-1 h) and phase specific localization in liver and lungs were greater with IgG(1-24 h) clearance rates for all the antibodies and IT-As used anti-6. This may be due to agglutination of tumor cells in the in these studies, as well as the two forms of ricin A chain, native peripheral blood and resultant trapping of the cell aggregates A and dgA. For all IT-As, two major phases of blood clearance in the lung and sinusoids of the liver. Nonspecific localization of 125I-NRtIg was 2-fold greater than 125I-ratFab' fragments in were observed: a very rapid initial phase (a phase) lasting approximately 1 h in which 60-95% of the IT-A was cleared both spleen and liver, suggesting that Fc binding contributes to nonspecific localization in these tissues. Fab' fragments of with a half-time (i./,)of 10-50 min; and a slower phase (hase) p in which the remaining 5-40% was cleared with a t</, f 4-22 h. NRtlg showed 7-8-fold higher localization in the kidneys, as o to These are consistent with the The results indicate that target cell binding plays a major role compared sites intact NRtlg. for IgGresultsFab' fragments (31-33). reported of clearance and in the f.. values for the a phase with antigen reactivity increasing The localization of IT-As prepared with native A chain is the initial rate of clearance from 2- to 5-fold. The form of the antibody (Fab' or IgG) or the A chain (native or dgA) had little shown in Fig. 4. Tumor-specific localization in spleens of the IgG-anti--l25I-Aand Fab'-anti-a-125I-A was less than that of effect on the f./,of the phase. In general, the hase clearance p the unconjugated antibodies (Fig. 3). In addition, nonspecific rate of all the IT-As was determined by the presence of A chain localization was as much as 5-fold higher in spleen and liver as (native A or dgA) and was essentially independent of the form of the antibody (Fab' or IgG) or its specificity (anti-5 or normal compared with unconjugated antibodies. Thus, the poor tumor immunoglobulin). Twenty-four h after injection, the blood lev localization observed with IT-As is probably due to their rapid els of the ITs prepared with dgA chain were consistently from removal from the circulation, largely by the liver. It is also 2- to 6-fold higher than the levels of the corresponding IT possible that recognition of A chain by reticuloendothelial cells in the spleen may impede the penetration of IT-As into the prepared with native A chain; however, with the single excep tumor tissue of this organ. The Fab'-IT-As were not cleared in tion of NRtlg-dgA, these values were less than 1% of the the kidney to the same extent as unconjugated Fab', perhaps injected dose so that the significance of these differences is due to their higher molecular weight (85,000 versus 55,000) questionable. Tissue Distribution and Tumor Localization of IT-As. In order and the rapid removal of IT-As from the circulation. As seen in Fig. 5, when specific and control antibodies were to assess the ability of the various forms of IT-A to localize coupled to dgA (instead of native A chain), the tumor-specific specifically in tumor tissue, the tissue distribution and tumor localization of the Fab'-anti-5-'25I-dgA and IgG-anti--'"I-dgA localization of the entire panel of antibodies and IT-As were determined at 10 min, 30 min, l h, and 4 h after injection. was significantly better than was observed with the same anti Preliminary experiments demonstrated that tissue levels of all bodies/fragments coupled to native A chain. In addition, non specific localization of the IT-dgAs was less than one-half of of these ITs reached maximum levels within 1 h of injection that observed with the native IT-As and was similar to that of and were decreasing by 4 h. BCLmice were used for these studies 3-4 weeks after i.p. injection of approximately 5 x 10* the unconjugated antibodies. Thus, the use of dgA decreased tumor cells. At this time, the large majority (>75%) of BCL, nonspecific tissue localization with a concomitant increase in Id+ tumor cells reside in the spleen which contains 1-2 x IO9 tumor-specific localization. Surprisingly, tumor-specific local ization of the Fab'-anti-6-dgA was approximately twice as high total cells and 4-10 x IO8Id* cells. In addition, a small number of leukemia cells (<I x 10" Id* cells/ml blood) are present in as for the IgG-anti--dgA. Possible reasons for the superior localization of the Fab'-dgA are discussed below. the circulation and variable numbers of tumor cells are present Breakdown of IT-As in the Circulation. In order to determine in the sinusoids of the liver (19). Normal IgD+ B-cells exist the relative stability of the Fab'-As and IgG-As in the circula primarily in the spleen and lymph nodes and are not present in tion, Fab'-anti-a-dgA and IgG-anti-6-dgA, which were essen either liver or lung tissue at sufficient levels for detection by these methods." A complete list of the tissues examined, as well tially free of nonconjugated antibody (less than 2% by silver staining of SDS-PAGE), were iodinated to label both the anti 4 R. J. Fulton fi al., unpublished data. body and A chain components. After i.v. injection, plasma
2621

per IgG molecule (M, 180,000-210,000).

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PHARMACOKINETICS OF IMMUNOTOXINS IN BCL,-BEARING MICE Table 3 Tissue localization ofanti-fi antibody versus anti-0-ITs

% of radioactivity injected OrganBladderEsophagusHeartLarge

intestineSmall intestineKidneysLiverLungsMesenteric

nodePeripheral lymph nodePancreasSalivarySkeletal lymph muscleSpleenStomachThymusThyroidUterus/ovariesBloodUrineLarge

contentsSmall intestine contentsStomach intestine contentsTotal

recoveryFab'-anti-60.080.020.050.160.473.675.741.440.640.280.100.182.4717.490.140.020.020.0815.262.370.070.540.2551.57Fab'-anti-a-A0.220.030.080.351.012.3716.431.141.190 " Values represent the mean of the percentage of injected cpm/organ at 15 min, 30 min, l h, and 4 h, determined in two mice for each protein at each time point. Percentages were calculated using total radioactivity in each organ. Variation of individual mice was less than 10% of the mean values.
dl Fob' onti-a mt Fab' Rat IgG IgG anti-fl Rat IgG

30 25 20 1510

30 25
20 is le s'

30

dl M

Fab' anti o-dgA Fab' Rat IgG-dgA

30

CD IgG anti S-dgA IgG-dgA Rat

tr O 2020

10-

10

50

Fig. 3. Tissue distribution of unconjugated antibodies in H( I , mice. The levels of '"I-labeled Fab' fragments (left) or IgG (right) of anti-5 antibody or NRtlg in spleen, liver, kidneys, and lungs were determined at 10 and 30 min and 1 and 4 h after injection into BCL, mice. Results are presented as the percentage of injected radioactivity per organ and represent the mean value of two mice per time point for each protein.

Fig. 5. Tissue distribution of IT-dgAs in BCL, mice. The levels of IT-'25I-dgA containing Fab' fragments (left) or IgG (right) of anti-e antibody or NRtlg were determined as described in Fig. 3. Results are presented as the percentage of injected radioactivity per organ and represent the mean value of two mice per time point for each protein.

40

CD Fob' anti -A M Fab' Rat iqG-A

40-,

dl IgG onti 6-A im Rat IgG-A

samples were obtained at intervals from 15 min to 48 h later and subjected to SDS-PAGE and autoradiography. Densitometric scanning of the autoradiographs allowed calculation of the relative amounts of l25I-IgG-A versus 125I-IgGand I25I-Fab'A versus I25l-Fab'. These values were corrected for the propor tion of radioactivity present in the antibody portion versus the A chain portion of the IT-As and for the differences in the clearance rates of IgG versus IgG-dgA or Fab' versus Fab'-dgA (see "Materials and Methods"). As shown in Fig. 6, the Fab':Fab'-dgA molar ratio decreased in a linear manner over the first 12 h and, by extrapolation, reached a value of 1.0 at 24 h. In contrast, the levels of IgG-dgA and IgG became equimolar by 8 h after injection. Thus, the Fab'-IT-As appeared to be 3-fold more stable than the IgG-IT-As in vivo. Binding Activity of IT-As Remaining in the Circulation. To determine whether the IT-As that remain in the circulation after the initial, rapid phase of clearance contribute to the binding of IT-As to target cells, the plasma samples obtained above were used in an in vitro binding assay with normal (surface IgD+) B-cells as target cells. As shown in Fig. 7, when compared

30

30-

20

20-

10-

Fig. 4. Tissue distribution of IT-As in BCL, mice. The levels of 1T-'"I-A containing Fab' fragments (left) or IgG (right) of anti-a antibody or NRtlg were determined as described in Fig. 3. Results are presented as the percentage of injected radioactivity per organ and represent the mean value of two mice per time point for each protein.

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PHARMACOKINETICS OF IMMUNOTOXINS IN BCL,-BEARING MICE

in the blood progressively lost antigen-binding activity; (d) specific tumor localization occurred rapidly and was essentially completed by 1 h; and (e) the highest degree of specific local ization to splenic tumor occurred when Fab'-anti-a-dgA was used. The presence of specific antigen markedly affected the a phase of clearance of the IT-As. The spleen contains the vast majority of tumor cells at 3-4 weeks after inoculation with BCL, cells (4-10 x IO8 Id+ cells/spleen). Thus, with anti-5-As, both Fab'-A and IgG-A were rapidly and, to some extent,
HOURS

Fig. 6. Stability of circulating IT-As. The Fab'-anti-o: Fab'-anti--dgA(O) or IgG-anti-a:IgG-anti-6-dgA () olar ratios in the circulation of mice was deter m mined by SDS-PAGE of plasma samples taken from 15 min to 24 h after i.v. injection of I25l-labeled proteins followed by densitometric scanning of autoradiographs of the gels. The relative amountss of antibody and IT-A were determined after correction of the densitometer values for the percentage of radioactivity present in the antibody versus A chain portion of the IT-As and for the different clearance rates of antibody versus IT-A as described in "Materials and Methods." Values represent the mean ratio obtained from two independent measurements in two mice for each IT-A (n = 4); bars, SD.

100

4812 HOURS

24

Fig. 7. Antigen-binding activity of circulating IT-As. The plasma samples obtained for Fig. 6 were assayed for antigen-binding activity using normal splenocytes, containing IgD* B cells as target cells (see "Materials and Methods"). The binding activity of the plasma samples is expressed as the percentage of activity in the iodinated preparations before injection. Fab'-anti-6-dgA ();gGI anti-3-dgA (O). Values represent the mean binding activity obtained from dupli cate assays of two different sets of plasma samples for each IT-A; bars, SD.

to the IT-As used for injection, the circulating IT-As lost approximately 80% of their initial antigen-binding activity by 10 h after injection. Since less than 10% of injected IT-A was present in the circulation at this time, the total antigen-binding capacity of circulating IT-As was less than 2% of that injected. These results indicate that both the IT-As and free antibody caused by agglutination of leukemic and/or normal B cells in which are present in the circulation during the phase of the circulation and their trapping in lung capillaries and liver clearance contribute minimally to tumor-specific binding and sinusoids, respectively. This explanation is consistent with the largely represent inactive IT-A or antibody. fact that marked infiltration of the liver parenchyma with BCL, cells does not occur until relatively late in the disease (19) and the lung is spared. DISCUSSION The fairly extensive localization of IT-A in muscle has not In this study we describe the pharmacokinetics of tumor- been reported in other studies of the localization of ITs (11reactive IT-As in tumor-bearing mice. An important feature of 13); however, significant localization to "carcass" (skin, skeletal the study is the use of both tumor-reactive antibody and control muscle, and bone) was reported in one study (13). The concen tration of IT-A in skeletal muscle was not sufficiently high to antibody which made possible quantification of the proportion of IT-A that specifically localizes to the tumor. The major allow a test of acid precipitability. Hence, we cannot formally findings are: (a) the initial a phase of blood clearance was very exclude the possibility that the radioactivity was free I25Ithat rapid and similar for the two anti-6 antibodies (Fab' and IgG) had dissociated from the IT-A. The finding is of interest in view and four anti-5-ITs regardless of the valency of antibody or of the clinical trials in which whole ricin (unconjugated to glycosylation status of the A chain. In contrast, Fab' and IgG antibodies) was administered to patients with cancer. One of NRtlg were cleared more slowly than the tumor-reactive anti the major symptoms in the patients treated with moderate or bodies, as were the ITs prepared from them; (b) Fab'-As dis maximally tolerated doses was muscle pain. This symptom sociated very slowly to free Fab' in the circulation whereas IgG- could be related to the above localization. As prepared with SPDP dissociated approximately 3 times Deglycosylation of the ricin A chain dramatically increased more rapidly; (c) intact IT-As and free antibody (IgG or Fab') the specific tumor localization of both Fab' and IgG ITs. As
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specifically localized to the spleen. Tumor cell binding was virtually complete within the first hour after injection, and less than 20% of the anti-5 IT-As remained in the circulation at this time. Thus, differences in the clearance of Fab'-A versus IgGA and between IT-As containing native or dgA chain were obscured by the presence of tumor which acted as an efficient antigenic sink. The tv, of the a phase of normal rat IT-As was 2-5 times longer than that of anti--IT-As reflecting the effect of specific localization to antigen-bearing cells. The remaining 10-15% of the injected radioactivity was cleared in the phase with a tv, of 4-12 h and probably did not play a significant role in localization of the anti-5-ITs to the tumor. This interpreta tion is supported by the similarity of clearance rates for specific and control ITs during the phase. The a and phase clearance rates reported in this study are similar to those reported by others (11-13). The only major discrepancy is the fairly rapid clearance of unconjugated normal IgG; however, the IgG used in these studies was rat IgG2a and IgG 2b and we are not aware of previous reports on the clearance of these subclasses of rat IgG in mice. The finding that during the phase of elimination of IT-A, biochemically intact IT-A in the blood gradually loses antigenbinding ability is a new observation. Hence, by 10 h after injection, even though IT-A and free antibody were present, there was little detectable antibody activity. We have considered three possible explanations for this finding: (a) IT-A and free antibody are denatured, e.g., by radiolabeling and purification; (b) the combining site is inactive because of mutations in the producer hybridoma cells; and (c) the antibody is bound to antigen (IgD), secreted or shed from BCL, cells or B-cells. There is no information to exclude any of these possibilities. The greater specific liver and lung localization of IgG-anti-5 compared to Fab'-anti-5 antibodies and their ITs was probably

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PHARMACOKINETICS OF IMMUNOTOXINS IN BCL,-BEARING MICE

shown previously, partial deglycosylation of ricin markedly decreases the localization of ricin (23) and IT-dgAs (13) to the liver, undoubtedly due to the important role of mannose recep tors on nonparenchymal cells. Thus, with less diversion to the liver, IT-dgA is able to recirculate and eventually gain access to the tumor cells in the spleen. The increased specific localization of Fab'-anti-<5-dgA com pared to IgG-anti-6-dgA is of potential importance for therapy. The factors responsible for this increased tumor localization are not clear. The major differences between the Fab'-dgA and IgG-dgA are: (a) the smaller size of the Fab' ITs (M, 82,000 for Fab'-A versus M, 180,000-210,000 for IgG-A); (b) lower avidity because of univalency; and (c) the different structure of the disulfide bond linking the antibody component to the A chain. Because of its smaller size, Fab'-A may be able to penetrate extravascular compartments of the spleen more read ily than IgG-A. In addition, modeling studies with radiolabeled antibodies suggest that penetration of antigen-expressing tis sues is more effective with a lower avidity antibody since "irre versible" binding to more accessible peripheral cells decreases the rate of diffusion into a tissue (33, 34). The major difference between the disulfide bonds in the Fab'A and IgG-A are the length of the bond and the site of the bond on the peptide backbone of the antibody molecule. The latter does not affect /';/ vitro antigen binding and is probably not related to differences in in vivo localization of the two types of conjugates. With regard to the length of the bond, SPDPmediated conjugation, used to prepare IgG-A, results in an interchain disulfide with a three-carbon spacer between the disulfide bond and the antibody molecule, as well as an addi tional four-carbon spacer in the case of (-amino substitution of lysine residues. The interchain disulfide in the Fab'-As contains only one carbon atom between the peptide backbone of either protein and the disulfide bond and, thus, would be less exposed. As reported previously by Blakey et al. (13) and confirmed in this study, the SPDP-IT-As are split in vivo so that free IgG appears in the circulation. In this regard, Fab'-As are more stable than the IgG-As. However, it is not clear that this relates solely to the disulfide bonds. This uncertainty is due to the complexity of the SDS-PAGE profiles of plasma, particularly for the IgG-As, and the fact that the activity of the immunoglobulin portion of the IT-A (antigen-specific versus control) affect the apparent rate of dissociation of the immunoglobulin component from the IT-A. Within 15 min after injection of the IgG-As, and to a lesser extent for the Fab'-As, a significant amount of high molecular weight material which was not pres ent in the injected preparation is observed on SDS-PAGE. The composition of this material has not been determined, but it is possible that IT-A undergoes disulfide exchange with one or more serum proteins. In addition, two prominent bands of lower molecular weight are generated from both IT-As, only one of which corresponds to the native antibody or Fab' frag ments. Thus, several catabolic mechanisms may contribute to the depletion of intact IT-A whereas only reduction of the disulfide bond will result in the appearance of native antibody or Fab' fragments on SDS-PAGE. In addition, preliminary data suggest that nonreactive IgG-NRtlg-dgA and Fab'-NRtlgdgA are both significantly more stable than the ITs prepared with anti-a.4 These results raise the possibility that reaction of the IT-A with target cells may contribute to the breakdown of the IT-A in vivo. Despite these considerations, the relevance of the "instability" of IT-A to tumor localization in this study is questionable due to the rapidity of tumor localization of the tumor-reactive IT-As. Since 80-90% of the anti-o-A is bound

to target cells within the first hour and the IT-As remaining in the blood lose the vast majority of antibody activity by 10 h, reduction and/or inactivation of IT-As in vivo is probably not sufficiently rapid to account for the more effective localization of the Fab'-anti-a-dgA compared to IgG-anti--dgA. In conclusion, there are several factors that could contribute to the superior tumor localization of the Fab'-anti-5-dgA. Re gardless of the interpretation, the increased localization of the Fab-anti-5-dgA predicts its effectiveness as a therapeutic reagent in vivo. This prediction is confirmed in the accompanying paper (35). ACKNOWLEDGMENTS
The authors thank N. Jasheway for technical assistance and Farrys Wood and Gerry Ann Cheek for secretarial assistance.

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