toxicity of a monoclonal f(ab')2: ricin a conjugate for ... · the f(ab')2 fragment of a...

(CANCER RESEARCH 44, 3178-3183, August 1984]

Toxicity of a Monoclonal F(ab')2: Ricin A Conjugate for Retinoblastoma

in Vitro1

John C. Merriam,2 Howard S. Lyon, and Devron H. Char3

Ocular Oncology Unit, Department of Ophthalmology and the Francis I. Proctor Foundation, University of California, San Francisco, San Francisco, California 94143

ABSTRACT

The toxic A chain of the poison ricin has been conjugated tothe F(ab')2 fragment of a retinoblastoma-specific murine mono

clonal antibody. The conjugate was toxic in vitro to Y-79 retino-blastoma cells at a concentration of approximately 5 x 10~10

M; Y-79 retinoblastoma cells were the immunizing cells in the

production of the monoclonal antibody. Against a second retinoblastoma cell line, the conjugate was toxic at a concentrationof approximately 10~9 M. Ricin A chain alone was not toxic toeither of these cell lines at concentrations less than 4 x 10~8 M.

The conjugate was no more toxic than was ricin A chainagainst five control cell lines in vitro.

The effect of the conjugate on retinoblastoma-derived cellswas concentration dependent. Concentrations less than a "minimum" concentration were not cytotoxic, despite an excess of

conjugate molecules over target cells. Concentrations greaterthan the minimum toxic concentration rapidly reached maximaltoxicity, suggesting that antigenic sites were saturated.

No clinical, hÃ©matologie,or renal toxicity was noted when micewere given injections of the conjugate at 1 mg/kg, or free ricin Achain at 0.375 mg/kg.

INTRODUCTION

Retinoblastoma is the most common malignant eye tumor ofchildhood. Tumor-related mortality is approximately 15% in de

veloped countries and significantly higher elsewhere (4). Contemporary treatment of tumor confined to the globe is highly successful, but most patients with metastatic disease rapidly die,despite chemotherapy and radiation.

Until recently, immunotherapy has been limited by the difficultyof producing antisera of appropriate specificity and avidity. Thecell hybridization techniques of Kohler and Milstein (13,14) havemade practical the production of monoclonal antitumor antibodies. Passive monoclonal serotherapy has shown promise inmurine tumor models; however, treatment was effective whengiven within 1 day of tumor inoculation (2, 11, 12). Using amonoclonal anti-idiotype antibody, Miller e? a/. (20) induced a

prolonged remission in a patient with nodular, poorly differentiated lymphocytic lymphoma. Six of 8 patients with cutaneousT-cell lymphoma had a partial response after murine anti-Leu-1

monoclonal antibody; none had a complete remission (17). Otherclinical trials of monoclonal serotherapy have not been success-

1This research was supported in part by an unrestricted grant from That Man

May See and by NIH Grants EY01441, EY01759, EY03675, and EY02162.2 Heed Foundation Fellow, and recipient of Training Grant EY-07058. Present

address: Edward S. Harkness Eye Institute, Columbia-Presbyterian Medical Center,

635 W. 165th St., New York, NY 10032.3 Robert McCormick Scholar of Research to Prevent Blindness, and recipient of

NIH Research Career Development Award K04-00117. To whom requests forreprints should be addressed, at Science 315, University of California, San Francisco, San Franciso, CA 94143.

Received July 27,1983; accepted April 25,1984.

ful. Treatment of a lymphoma (21), a T-cell leukemia (19), chronic

lymphocytic leukemia (8), and acute lymphoblastic leukemia (30),resulted in a transient reduction in circulating tumor cells, but nochange in clinical course. No therapeutic response was noted in2 patients with metastatic melanoma after administration ofmurine monoclonal antibody (31). While optimal doses remain tobe determined, these cases demonstrate that in most clinicalsituations, antibodies alone may not be therapeutic.

The conjugation of a monoclonal antibody to a toxin such asricin creates a potentially more potent Â¡mmunotherapeutic agent,and in vitro studies of such conjugates generally have confirmedtheir toxicity (3, 7, 9,10, 15, 16, 22, 25, 33, 35). Ricin, a poisonderived from the castor bean, has a number of potential advantages as a toxin for immunotherapy. Ricin molecules consist of2 polypeptide chains linked by a disulfide bond; the B chainfunctions as a carrier and the A chain functions as a toxin.Separation of the 2 chains results in no loss in activity of the Achain. While no A chain:antibody conjugate is as toxic as intactricin, substitution of the monoclonal antibody for the B chainshould make the conjugate relatively tumor specific. Ricin is toxicto both dividing and nondividing cells. The A chain catalyticallyinhibits protein synthesis, and theoretically, as few as one or 2A chain molecules in the cytosol could kill a cell (23).

We have previously described a murine monoclonal antibodyto human retinoblastoma-associated antigens (5). This reportdescribes the conjugation of the F(ab')2 portion of this antibody

to the A chain of ricin and demonstrates its toxicity to tissueculture cell lines derived from retinoblastoma.

MATERIALS AND METHODS

IgG Purification and Conjugation to Ricin A Chain. The antibody(P38) was produced by the immunization of BALB/c mice with Y-79

retinoblastoma cells, followed by the hybridization of immune spleencells with murine myeloma cells (5). At appropriate steps in the preparation of the conjugate, purity and specificity were assessed with Ouch-terlony's double-diffusion technique and an ELISA (5).4 Protein concen

tration was determined with the Lowry method, or by comparing absorb-

ance at 280 nm with a reference standard. The 2 methods gave similarresults.

BALB/c mice were given injections i.p. of 0.5 ml pristane (2,6,10,14-

tetramethylpentadecane) (Sigma Chemical Co., St. Louis, MO), 2 to 5days prior to the injection of 0.5 ml of a suspension of hybridoma cells(106 cells/ml). Approximately 2 weeks later, the ascites was harvested

and pooled from groups of 5 or 6 mice. Monoclonal IgG was separatedfrom whole ascites by passage on a DEAE-Affi-Gel blue column (Bio-

Rad Laboratories, Richmond, CA), or by ammonium sulfate precipitationand passage of reconstituted immunoglobulin over a Sephacryl S-200

column (Pharmacia Fine Chemicals, Piscataway, NJ). The IgG fractionswere pooled, concentrated, and enzymatically cleaved with pepsin(Sigma) at a ratio of 3 mg enzyme/100 mg protein for 18 to 24 hr. The

4The abbreviations used are: ELISA, enzyme-linked Â¡mmunosorbentassay;PBS, phosphate-bufferedsaline.

3178 CANCER RESEARCH VOL. 44

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Toxicity of F(ab')2:Ricin A Conjugate

digested Â¡mmunoglobulin was filtered on a Sephacryl S-200 column, and

the purity was checked with 12.5% polyacrylamide gel electrophoresis.The F(ab')Â¡>fragments were dialyzed and concentrated against 0.01 M

PBS (Grand Island Biological Co., Grand Island, NY) to a minimumconcentration of 0.5 mg/ml.

F(ab')s fragments were derivatized with a 30-fold excess of A/-SUC-

cinimidyl-3-(2-pyridyldithio)propionate (Pharmacia). Approximately 2 to 3mol of A/-succinimidyl-3-(2-pyridyldithio)propionate were attached to each

mol of antibody fragment. The mixture was dialyzed twice against 1 literof PBS.

Ricin A chain (EY Laboratories, San Mateo, CA) was dialyzed againstsodium acetate buffer (pH 5.5) with 0.01 M cysteine, and sulfhydrylgroups were reduced with dithiothreitol solution (Sigma). This mixturewas passed over a Sephadex G-25 (Pharmacia) column and centrifugea

to remove precipitate.A 2-fold molar excess of activated ricin A chain was incubated over

night with derivatized Ffab'fe at room temperature. The solution was

concentrated before passage over a Sephacryl S-200 column to remove

free A chain and uncoupled antibody. Polyacrylamide gel electrophoresisrevealed that 1 to 3 ricin A chain molecules were attached per F(ab')?

fragment. Most antibody fragments had 2 ricin A chains attached.Calculation of Molarity. To calculate the molarity of the purified

conjugate, it was assumed that all the protein in solution was conjugate,and that each conjugate had 2 ricin A chain molecules attached (M,170,000). The calculation of molarity of ricin A chain was based on amolecular weight of 30,000.

Tissue Culture Cell Lines. The Y-79 retinoblastoma cell line has been

described previously and has been maintained in our laboratory forapproximately 10 years (28).

Lemmon's retinoblastoma cells were derived from a Caucasian male

with a sporadic somatic unilateral retinoblastoma. It was maintainedinitially on a female human fibroblast feeder layer until it became self-

supporting as a nonadherent line in Roswell Park Memorial InstituteMedium 1640 with 10% fetal calf serum. The HLA types of the cell lineand the patient were identical. No cytogenetic abnormalities were notedon Giemsa chromosome analysis. Isoenzyme mobility was consistentwith a human cell line.

Ultrastructural examination of both retinoblastoma-derived cell lines

revealed features of retinoblastoma: surface blebs, microtubules, roughendoplasmic reticulum, secretory granules, and ribosomal aggregates.Nuclei had clumped chromatin, nuclear infoldings with triple membranes,and prominent nucleoli (6).

The Raji cells were a gift of A. N. Theofilopoulos (32). The other celllines were the gifts of various investigators, and have been maintainedin our laboratory for many years.

Cytotoxicity of the Conjugate. A trypan blue exclusion assay wasused to assess the toxicity of the conjugate and control sera for 2retinoblastoma cell lines and 5 control cell lines; 0.5 ml of a suspensionof the target cells at an approximate concentration of 2 x 105/ml was

transferred to 24-well tissue culture plates (Costar, Cambridge, MA). The

appropriate amount of conjugate, control solution, or medium (RoswellPark Memorial Institute Medium 1640 with 10% heat-inactivated fetalcalf serum, glutamine, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic

acid, penicillin and streptomycin, and Fungizone) was then added tobring the total volume to 1.0 ml. A small aliquot (15 Â¿J)of each well wasremoved periodically to determine both total cell number and cell viabilitywith trypan blue. Assays were terminated either after 7 days, or whenthe rate of growth of cells in medium alone slowed, due to cell crowdingand depletion of the medium. As additional controls, cells were incubatedwith varying concentrations of unconjugated whole antibody, ricin Achain, or free antibody and free ricin A chain.

All experiments were performed in duplicate or triplicate; most assayswere repeated to confirm significant differences.

Statistical Analysis. All calculations were made with the natural log(In) of the number of viable cells. Student's i test was used to comparethe number of viable cells in 2 different media. Wilcoxon's rank-sum

statistic was used to compare the rates of growth of cells in 2 different

media. The null hypothesis was that "treatment" (e.g., conjugate at a

specific concentration) had no effect in comparison with the control. Forboth tests the significance level was 5%.

As Student's i test compared cell numbers at a particular time, its

validity might be compromised by a difference in the initial number ofcells in the test populations. Wilcoxon's rank-sum statistic compared the

rate of growth of 2 test populations over the entire course of the assay,and should be independent of this variable. The 2 methods led to rejectionof the null hypothesis at the same test concentrations in most cases;when they differed, the Wilcoxon test was more stringent; these dataare summarized in Table 1.

Toxicity of Conjugate in Vivo. Groups of 4 male BALB/c mice weregiven injections i.v. of 250 Â¡i\of one of the 4 test solutions: conjugate (1mg/k9). free ricin A chain (0.375 mg/kg), free anti-retinoblastoma mon

oclonal antibody (1 mg/kg), and PBS. As each conjugate molecule hadapproximately 2 ricin A chains attached, a dose of ricin A chain equivalentto twice the molar amount of conjugate was used. The dose of wholemonoclonal antibody (monoclonal P16 with specificities similar to thoseof monoclonal P38 was used) was adjusted to molar equivalency withthe dose of conjugate.

The animals were checked daily for 1 week for clinical signs of toxicity,and thereafter were checked twice weekly for 2 months. The growthinhibitory effect of the conjugate's was not significant in vitro until 5 to 7

days after inoculation. Therefore, 5 days after i.v. injection, blood wasobtained from the retrobulbar plexus of each mouse for determination ofhematocrit, blood urea nitrogen, and creatinine.

RESULTS

ELISA

The P38 monoclonal antibody showed slightly less activitythan a polyclonal serum against 2 retinoblastoma-derived tissueculture cell lines (Y-79 and Lemmon's). The P38 monoclonal

antibody showed no cross-reactivity with 12 control cell lines

(including all cell lines tested in the cytotoxicity assays below),except for minimal reactivity with Raji cells. As the antibody wascleaved and conjugated, its activity in the ELISA decreased.

Cytotoxicity

P38 monoclonal antibody alone had no inhibitory effect on cellgrowth. The addition of free monoclonal antibody to free ricin Achain neither increased nor decreased the toxicity of ricin A chain(data not shown). All assays were performed with the second

Table 1Summary of Wilcoxon rank-sum statistic

ConjugateCell

lineY-79

retinoblastomaLemmons retinoblas

tomaMelanoma 157Neuroblastoma IMR5Neuroblastoma B. JonesRajiHuman peripheral blood

lymphocytesConcen

tration"(M)5

x 10-105x10-Â«1

x UT"4x 10-"04 x 10-**

None1 x 10-"P0.0017

0.0130.0076

0.01430.00110.033Ricin

AchainConcen

tration(M)4X10-"

1 x1Q-71

x 10-*1 x 10-'1 x 10""

None1 x 10-7P0.0047

0.00110.0022

0.01430.00110.95Minimum

[ricin]:min-imum [con-

jugatef80

2010.25

0.25

Minimum concentration of conjugate at which a significant inhibition of cellgrowth was noted, using Wilcoxon rank-sum statistic.

Ratio of minimum toxic concentration of ricin A chain to minimum toxicconcentration of conjugate.

cAt1 x 10-"M, p = 0.0571.dAM x 10-Â°M,p = 0.30.

AUGUST 1984 3179



J. C. Merriam et al.

batch of conjugate (Conjugate 2) prepared in the laboratory.Y-79 Retinoblastoma Target Cells. Y-79 cells grew rapidly in

normal medium (Charts 1 and 2). The effect of both ricin A chainand the conjugate was concentration dependent. For both freeA chain and the conjugate, concentrations greater than theminimum inhibitory concentration appeared to have an increasinginhibitory effect on cell growth, up to a concentration at which amaximum rate of cell death occurred.

The conjugate had a marked inhibitory effect up to a concentration of approximately 5 x 10~10M (Chart 1; Table 1). Ricin Achain alone had a significant inhibitory effect only to 4 x 10"8 M

(Chart 2). Comparison of the minimum effective concentrationsof the conjugate and ricin A chain shows that the conjugate wasapproximately 80 times more potent against Y-79 cells than was

ricin A chain (Table 1).Lemmon's Retinoblastoma Cells. The F(ab')2:ricin A chain

conjugate was less effective against this cell line than against Y-79 cells. Conjugate concentrations of 4 x 10"9 M or greater werecompletely cytotoxic; at 10"9 M the conjugate delayed, but did

not prevent cell growth (Chart 3).In contrast, ricin A chain was cytotoxic only at 10~7 M. At 4 x

10~B M, ricin A chain delayed, but did not prevent cell growth

(Chart 4).The conjugate was approximately 20 times more potent than

was ricin A chain against Lemmon's retinoblastoma (Table 1).

Melanoma 157 Cells. Charts 5 and 6 demonstrate that ricinA chain and the conjugate were nearly equally toxic to this

2.5X106

2.0X106

Ã•3 1.5X106

DU

fcOC

Coni 10~8M

Conj 1(T9Mâ€¢~Â°â„¢Â°â„¢Conj 5x10'10M

- â€”â€”â€”â€”Conj 1CT10M

IO1

500,000

CONJUGATE No. 2 vs.Y-79

J_ JDAY 1 3 5

Chart 1. Effect of various concentrations of the Ffab'^ricin A chain conjugateon the growth of Y-79 retinoblastoma cells in vitro. Approximately 105 Y-79 cells

were incubated with each test concentration on Day 0.

i8u_o

5z

2.5X106r-

2.0X106

1.5X106-

500,000 -

O -

Ricin A 10'7M

Ricin A 7x10~8M

Ricin A 4x10"8M

- Ric,nA 10-8MRicin A 5x10"9M

Ricin A KT9M

DAY 1Chart 2. Effect of various concentrations of ricin A chain on the growth of Y-79

retinoblastoma cells in vitro. Approximately 105 cells were incubated with each test

concentration on Day 0.

control cell line. At a concentration of 10~8 M or greater, both

resulted in killing of all target cells.Neuroblastoma IMR5 Cells. The conjugate was toxic only at

a concentration of approximatley 4 x 10~8 M or greater. Ricin Achain was toxic only at concentrations greater than 10~8 M(Table

1).Neuroblastoma B. Jones Cells. Charts 7 and 8 show that

ricin A chain was slightly more toxic to this control cell line thanwas the conjugate (Table 1).

Human Peripheral Blood Lymphocytes. Peripheral blood lymphocytes from 2 healthy volunteers were pooled. The cellsshowed a progressive decline in number in culture, and neitherthe conjugate nor ricin A chain was notably toxic (Table 1). Theconjugate had a growth-inhibitory effect only at 10~8 M. Ricin Achain had no effect on growth up to 10~7 M.

Raji Cells. The Raji cell was the only control cell line to whichP38 monoclonal antibody demonstrated any binding in theELISA. In these experiments, neither ricin A chain nor the conjugate had any significant inhibitory effect on the rate of growthof Raji cells (Table 1).

Toxicity of Conjugate in Vivo

No effect on hematocrit, blood urea nitrogen, or creatinine wasnoted 5 days after the i.v. injection of conjugate (1 mg/kg), freericin A chain (0.375 mg/kg), or whole P38 monoclonal antibodyantibody (1 mg/kg), relative to control injection of PBS. No grossclinical effects were noted up to 2 months after injection.




Toxicity of F(ab')2:Rlcin A Conjugate

8u.OC

2

3.5X106

3.0X106

2.5X106

2.0X106

1.5X106

10e

CONJUGATE No.2vs.LEMMONS RB

Conj 4x10'8lv1

Conj 1Cr8M

Conj. 10"9M

nj. 5x1Cr10M

._._.... Conj. 1CT10M

500,000

0 -

DAY 1357Charts. Effect of various concentrations of the F(ab')j:ricin A chain conjugate

on the growth of Lemmon's retinoblastoma (RB) cells in vitro. Approximately 105

cells were incubated with each test concentration on Day 0.

3.5X10V

3.0X106

2.5X106

2.0X106

1.5X106

10'

500,000

0 -

RICIN A vs. LEMMON S RB-^^â€” Ricin A 10"7M

Ricin A 4x1CT8M

RicinAKr8MRicin A 5x10"9MRicinA10'9M

DAY 1357Chart 4. Effect of various concentrations of nein A chain on the growth of

Lemmon's retinoblastoma (RB) cells in vitro. Approximately 105cells were incubated

with each test concentration on Day 0.

10'

yj 700,000

8UuOocLUcoÂ§ 300,0002

O -

CONJUGATE No.2 vs. MELANOMA No.157

Conj 4xKT8M

Conj Kr8MConj. 1Cr9MConj 10-1Â°M

Conj. 1CT11M

DAY 1 3 5 7Chart 5. Effect of various concentrations of F(ab')j:ricin A chain conjugate on

the growth of melanoma 157 cells in vitro. Approximately 105 cells were incubated

with each test concentration on Day 0.

IO6

700,000

UJuu.OocLUco

300,000

O -

RICIN Avs MELANOMA No 157Ricin A 10~7M

Ricin A 10'8M

Ricin A 10"9M

Ricin A 10"10M

RicmA10"11M

A

DAY 1Charte. Effect of various concentrations of ricin A chain on the growth of

melanoma 157 cells in vitro. Approximately 10s cells were incubated with each test

concentration on Day 0.

DISCUSSION

We have conjugated the A chain of ricin to the F(ab')2fragmentof a murine monoclonal anti-retinoblastoma antibody, and haveshown that this conjugate is both toxic and relatively specific forretinoblastoma cells.

The activity of the intact monoclonal antibody in the ELISAappeared to reflect the toxicity of the conjugate for target cells.The monoclonal antibody was more active in the ELISA againstY-79 cells than against Lemmon's retinoblastoma cells; the

conjugate proved to be more toxic to Y-79 cells than to Lemmon's cells. Against the control cell lines to which the antibody

showed no activity in the ELISA, the conjugate was no moretoxic than ricin A chain alone. Although the P38 antibody wasweakly reactive with Raji cells in the ELISA, the conjugate didnot prove to be more toxic than ricin A chain alone to this cell.

Studies with other tumor lines have shown the importance ofconjugate concentration to cytotoxicity (9, 18, 26), and these

AUGUST 1984 3181



J. C. Merriam et al.

500,000

Ou.OocLUco

300.000

100,000

o _

CONJUGATE No 2 vs. B JONES

Con) 4x10"8M .

Conj10"8MConj 10"9M

â€” Con) 10"10M _.-â€¢'

Con) 5x1011M.-''

J_

DAY 1357Chart 7. Effect of various concentrations of the Ffab'feiricin A chain conjugate

on the growth of neuroblastoma B. Jones cells in vitro. Approximatelywere incubated with each test concentration on Day 0.

cells

500.000

E

ILu 300.000OLUOOCLUOD

2Z>z 100,000

RICIN A vs. B JONES

Ricin A 10"7

Ricin A 10"8

Ricin AIO"9

â€”_â€” R,cin A 10~10- Ricin A10"11

DAY 1 3 5 7Charts. Effect of various concentrations of ricin A chain on the growth of

neuroblastoma B. Jones cells in vitro. Approximately 105cells were incubated witheach test concentration on Day 0.

experiments confirm this. At successive dilutions of 10~", 10~9,and 5x10"10 M, the efficacy of the conjugate against Y-79 cells

was only slightly reduced, suggesting that saturation of availableantigenic sites had been reached. Further reduction to 10~10M

markedly decreased the toxicity of the conjugate, although at10~10 M, conjugate molecules are present in excess of targetcells (approximately 60,000:1, calculated with Avogadro's number). With Lemmon's retinoblastoma cells, concentrations of theconjugate of 5 x 10~9 M or greater led to complete lysis of alltarget cells. At a concentration of 10~9 M, cell growth appeared

only to be slowed. At lower concentrations, no effect on growthof Lemmon's retinoblastoma was seen.

Although the conjugate was more potent against Y-79 cellsthan against Lemmon's retinoblastoma, the toxicity of free ricinA chain appeared to be similar for these 2 cell lines. Lemmon's

retinoblastoma cells thus did not appear to be intrinsically more

resistant to the toxin. Rather, both the decrease in reactivity inthe ELISA and the decreased potency of the conjugate suggestthat the P38 monoclonal antibody recognizes fewer binding siteson Lemmon's cells than on Y-79 cells, binds to these cells less

avidly, or both. Raso ef al. (26) also found variable toxicity of amonoclonal F(ab')s:ricin A conjugate to a number of leukemia

cell lines.Parallel effects of dilution were also observed with ricin A

chain, although at higher concentrations. The target cell-specificF(ab')2 carrier clearly facilitated the entry of a toxic dose of ricin

A chain at a lower concentration than when only free A chainwas present. Even at maximum concentration, neither ricin Achain nor the conjugate killed all target cells until at least 3 to 5days after exposure to the conjugate. Other investigators havealso shown a gradual decline of target cell numbers (18, 25, 26),or of protein synthesis (22), with toxic concentrations of anantibody:ricin A chain conjugate. The lag in protein synthesisinhibition and cell death may be due primarily to a delay in entryof the toxin into the cytoplasm (24, 27).

The clinical efficacy of a monoclonal antibody:toxin conjugatedepends on many variables, but of paramount importance is theexpression of shared tumor-associated antigens on all malignant

cells. The heterogeneity of cell surface antigens within a singlehost is a problem of unknown significance. Badger and Bernstein(1) found that progressive disease in leukemic mice treated witha monoclonal antibody was due to the development of stableantigen-negative cells. Uyttenhove et al. (34) showed that mÃ©tas

tases of a mouse mastocytoma may escape rejection becauseof loss of surface antigens.

Both free monoclonal antibody as well as monoclonalF(ab')2:ricin A chain conjugate have been shown to induce mod

ulation of the common acute lymphoblastic leukemia antigen (26,29,30). Treatment of a patient with a T-cell leukemia with murinemonoclonal antibody to a T-cell differentiation antigen (Thy-1.1)

induced modulation of this target antigen (19). It is not known ifprimary or metastatic retinoblastoma is capable of antigenicmodulation.

Another potential problem with an antibody:toxin conjugate isnonspecific toxicity. Against control cells to which the antibodyshowed no activity in the ELISA, the conjugate was no moretoxic than was ricin A chain. The limited animal data reportedhere and elsewhere indicate that antibody:toxin conjugates aretolerated well (26). However, further studies in vivo need to bedone to determine the safety and efficacy of antibody:toxinconjugates.

ACKNOWLEDGMENTS

The authors are grateful to Professor Kenneth WÃ¤chterof the Departments ofDemographyand Statistics, Universityof Californiaat Berkeley, for his advice andhelp with the statistical analyses.We would also like to thank Joan Weddell for thepreparation of the charts, and Kimberiy Huhta and Anna Kaminski for technicalassistance.

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Toxicity of F(ab')2:Ricin A Conjugate

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AUGUST 1984 3183



1984;44:3178-3183. Cancer Res John C. Merriam, Howard S. Lyon and Devron H. Char

in VitroRetinoblastoma :Ricin A Conjugate for2)′Toxicity of a Monoclonal F(ab

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