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The Use of Monoclonal Antibody G250 in the Therapyof Renal-Cell Carcinoma

E. Oosterwijk, F.M.J. Debruyne, and J.A, Schalken

S INCE KOHLER AND MILSTEIN described

the technique to isolate monoclonal antibod­

ies (MoAbs),1 much effort has been focused to

isolate MoAbs reactive with tumor antigens for

diagnostic and therapeutic applications.2'0 Despite

intense research, the isolation of clinically relevant

MoAbs has been disappointing. Preferably, such

MoAbs are reactive with tumor-specific antigens

(TSA) which should be expressed by all tumor cells

of a certain tumor type. However, unequivocal

evidence for unique TSA in human malignancies is

still lacking. Most human malignancies have weak,

if any, detectable immunogenic properties, indicat-

ing that most human cancers either are nonimmu-

nogenic or mediate strong immunomodulatory ef­

fects. Current MoAbs of interest recognize tumor-

associated antigens (TAA) which are either

differentiation antigens (transiently) expressed dur- ing organogenesis, or aberrantly expressed anti­

gens, which are (transiently) expressed elsewhere

in nonrelated normal tissue(s).

Administration of MoAbs that react with an

antigen present on many normal tissues may result

in unwanted side effects and diminish the amount

of MoAb delivered to the tumor, influencing the

efficacy of MoAb treatment. Therefore, 1) little

cross-reactivity with nontumorous tissues is prefer­

able; 2) the TA A density on the tumor cells should

be high to achieve sufficient accumulation of

MoAb; and 3) all tumor cells must express the

TAA to enable the MoAb to specifically target to

all tumor sites, eg, to accomplish visualization of all

tumor sites or to achieve complete tumor remis­

sion. Unfortunately, for the vast majority of TAA

that have been defined by anticarcinoma MoAbs,

these criteria are not met: 1) the TAA is usually

expressed on more than one type of carcinoma; 2)

not all tumors within a given tumor type express

the TAA (intertumor heterogeneity); 3) not all

From the Departm ent of Urology, A cad e m ic Hospital Nijmegen, T/ie Net/ier/ands.

Address reprint requests to E. Oosterwijk, P h D , Urological

Research Laboratory, Department of U ro logy , A cad e m ic Hos])itai Nÿmegen, Geert Grootepicin Zuid. 16, PO B o x 9 10 1 , 6 5 0 0 H ß Nÿmegen, T h e Netherlands.

Copyright © J 995 by W .B . Saunders Company 0 0 9 3 ~ 7 7 5 4 f 9 5 / 2 2 0 l '0 0 0 8 $ 0 5 . 0 0 / 0

cells within a tumor lesion, including métastasés,

express a particular TAA (intratumor heterogene­

ity); 4) lesions of one patient express a TAA to a

varying degree; 5) temporal modulation of TAA

may occur; and 6) TAA expressed on most cells of

a particular carcinoma are often expressed on some

normal adult tissues. In general, with higher percent­

age of positive tumor cells and tumors expressing

the TAA, cross-reactivity with normal tissues in­

creases.

MOABS REACTIVE WITH RENAL-CELLCARCINOMA

A number of MoAbs reactive with cell surface

antigens of renal-cell carcinoma (RCC) have been

identified.7'15 The specificity of the majority of

RCC-related MoAbs has been established using

immunohistochemistry. Less is known about their

targeting abilities and antitumor efficacy. The

MoAbs can be divided into two groups: 1) MoAbs

recognizing differentiation antigens,7'14 and 2)

MoAbs recognizing RCC-TAA aberrantly ex­

pressed in RCC, ie, recognizing an RCC-antigen

absent from normal kidney.8'10,14,15 Most MoAbs of

the latter category show very restricted cross­

reactivity with normal tissues as judged using

immunohistochemistry. It appears that these MoAbs

identify different RCC-TAA because they display

different cross-reactivity with normal tissues. In

view of their restricted cross-reactivity with normal

tissues in combination with expression of a given

RCC-TAA in most RCC, these MoAbs are prime

candidates for clinical investigations.

One of the MoAbs recognizing an aberrantly

expressed RCC-TAA, MoAb G250, was obtained

after fusion of spleen cells from a mouse immunized

with fresh RCC homogenates.15 This MoAb identi­

fies an antigen absent from normal kidney and

other normal tissues examined, with the exception

of gastric mucosal cells and cells of the larger bile

ducts. In the initial study, G250 antigen expression

of 55 RCC was examined: 42 of 47 primary RCC

showed homogeneous G250 antigen expression

(89%), four tumors showed heterogeneous G250antigen expression and only one primary tumor was

completely devoid of G250 antigen expression.

More importantly, of eight métastasés examined,

34 Sem/ntfrs in Oncology, Voi 22, No I (February), 1995: pp 34-41

MoAb G250 IN RENAL CANCER 35

five showed homogeneous G250 expression (62%),

two showed heterogeneous U250 expression and

one metastasis failed to express U250 antigen.b

With an increased number of RCC tested, no

difiere nee with respect to the percentage of RCC

expressing G250 antigen has been observed. In 77

of 95 (81%) primary RCC cases that were investi­

gated, more than 50% of tumor cells were scored

positive and in an additional eight patients, primary

tumor cells also stained, albeit with lower fre­

quency; ie, approximately 90% of primary RCC

express C250 antigen. For the metastatic lesions,

G250-positive tumor cells were noticed in 18 of 22

tumors (82%) examined, with homogeneous stain­

ing in 9 of 17 metastatic RCC examined (53%). in

general, clear cell RCC tend to show homogenous

G250 antigen expression, whereas non-clear cell

RCC show heterogenous G250 expression. In view

of the restricted cross-reactivity with normal tissues

and the homogeneous expression in most RCC

examined, this MoAb seemed useful as a therapeu­

tic and/or radiodiagnostic agent,

EXPERIMENTAL DATA

Tumor Studies

The preferential accumulation of MoAb G250 was investigated in a RCC xenograft model.16

BALB/c nu/nu mice were xenogrufted with human

RCC and/or human noivRCC tumors, and in­

jected with 12̂ I-labeled MoAb G250 lgGl, F(ab')2

or Fab'. Specific MoAb G250 accumulation was

observed in the RCC tumors for all antibody forms.

G250-negative tumors did not show any MoAb

G250 uptake, nor did the G250-positive tumors

show increased uptake of nonrelevant immuno­

globulin. Higher uptake was generally found with

intact G250 than with G250 F(ab')¿ or Fab'

fragments, most probably because of slower blood

clearance, leaving the antibody more time to diffuse

to the tumor cells. The lowest uptake was observed

for Fab' fragments, related to a short biological

half-life and lower avidity. Similar studies were

performed by Chiou et al with MoAb A6H, an

MoAb that reacts with a normal kidney-difierentia-

tion antigen.17 For MoAb G250 as well as MoAb

A6H relatively high tu mor/blood ratios were ob­

served compared with other mouse targeting stud­

ies. This is possibly related to a general increase in

permeability of the vascular bed in RCC xeno­

grafts.18

In addition to these mouse-targeting studies,

supplementary studies were performed with ex vivo

perfused tu mor-bearing human kidneys.19 In this

model system tumor-bearing kidneys are flushed

with preservation fluid immediately after surgery,

whereupon the kidneys can be perfused with the

MoAb of interest. Using this model system, the

tumor targeting ability of 99mTc-labeled MoAb

G250 was investigated. yy,nTc MoAb G250 imaging

of tumor-bearing kidneys resulted in clear images of

RCC, with no MoAb G250 uptake in normal renal

tissue. Despite the low temperature (0 to 4°C to

assure appropriate pressures) and the relatively

short circulation time (16 hours), tumor to kidney

ratios were approximately 8:1.19 The preferential

uptake of MoAb G250 in these ex vivo experi­

ments were a clear indication that this MoAb held

promise for clinical use.

MoAb G250 Mediated ThcraljyTumor cells may be lysed by MoAb-directed

effector cells, so-called antibody-dependent cell-

mediated cytotoxicity (ADCC). Several cell types

have been shown to be active in ADCC, including

macrophages, natural killer (NK) cells, killer (K)

cells, and neutrophils. Experimental studies have

highlighted the importance of antibody subclass»20

and mouse MoAbs of the IgG2a subclass have been

shown to have the greatest activity in ADCC

a si'* ; 21.22

The antitumor effect of MoAb 0250 alone and

in combination with biological response modifiers

(BRMs) was tested in a nude mouse model.23

Because the original MoAb G250 clone produced

immunoglobulin of lgGl subclass, a poor mediator

of ADCC, an IgG2a class switch variant of the

original clone was developed. As a consequence,

MoAb G250 specificity, idiotype and avidity were

retained. Mice carrying established subcutaneous

RCC tumors of approximately 50 mm* were treated

with different doses. Administration of 100 pug

MoAb G250 per mouse for 6 weeks with three

weekly injections resulted in a significant (P < .01)

inhibition of mean tumor growth to 50% of control

tumors. Increasing the MoAb G25Ö dose to 500

fJLg/dose, showed an inhibition of mean tumor

growth to 35% of controls (P < .0001 versus

control group). However, this increased tumor

growth inhibition was not statistically different

from the 100-|xg dose schedule. No complete

tumor regression was observed in any of the treated

animals.

36 OOSTERWIJK, DeBRUYNE, AND SCHALKEN

For this RCC xenograft (NU12), the optimal

treatment schedule with biological response modifi­

ers had been established in earlier studies.24 BRM

treatment alone resulted in significant tumor growth

inhibition, but complete tumor regression was

never observed, despite the intense treatment sched­

ule. We combined the best BRM treatment, rIFNa

plus rTNFa, with MoAb G250 treatment to inves-

tigate whether the efficacy of this combined treat'

ment was superior to either treatment alone. This

combination treatment significantly reduced NU12

tumor growth as compared with MoAb G250 or rIFNa/rTNFa alone (P < .0006 versus MoAb

G250 alone, P < .003 versus rlFNa plus rTNFa).

In the majority of mice tumors stabilized, and in

several mice complete tumor regression was ob-

served. No effects were observed when mice were

treated with irrelevant IgG or with MoAb G250

F (ab') 2 fragments, indicating that the effect was

MoAb G250 specific and Fc-dependent. The Fc-

dependency also indicated that ADCC was prob'

ably playing a role in the destruction of tumor cells.

Histochemical examination revealed that MoAb

G250- or rlFNa/rTNFa-treated tumors were infil­

trated by very few macrophages, if any, similar to

control tumors in which macrophage infiltrates

were absent. In contrast, massive macrophage tu­

mor infiltration was observed in MoAb G250/

rIFNa/rTNFa-treated tumors. These infiltrates

seemed to be infiltrating viable tumor nests and

surrounded large necrotic areas. Most likely, BRM

treatment leads to activation of mouse NIK cells

and macrophages, both mediators of ADCC, result­

ing in effective macrophage-mediated ADCC.23

Although these results are encouraging! it re­

mains to be investigated whether unmodified MoAb

G250 IgG2a will be useful therapeutically. We have

performed in vitro ADCC experiments using fresh

isolated human monocytes as effector cells, and

have not observed any tumor cell lysis. Different

RCC cell lines were used to circumvent problems

that may occur because of the use of a lysis -

resistent cell line, and to assure that antigen densi ty

(which has been shown to be important in active

ADCC,25) did not play a role. MoAb G250 IgG2a

may not be effectively recognized by these human

effector cells or, alternatively, these effector cells

may have to be activated to mediate ADCC or

G250-antigen density is too low. The latter possibil­

ity seems unlikely in view of the high number of

antibody binding sites on these RCC cell lines.9

Enhancement of the ADCC capability of MoAb

G250 can probably be achieved by chimerization of

the antibody (chimeric MoAbs consist of mouse Ig

variable regions grafted onto human Ig constant

regions). However, preliminary ADCC experi­

ments with chimeric G250 lgGl and purified

human monocytes as effector cells have been

unsuccessful. Combining activated human lympho­

cytes with chimeric MoAb G250 may facilitate

ADCC.

CLINICAL EXPERIENCE

Significant clinical experience with radiolabeled

murine MoAbs that detect TAA has been acquired

in the last decade.2'6 Radioimmunoscintigraphy

studies of RCC with MoAbs have been limited,

generally restricted to animal models.16,17,26'29 Ves-

sella et al performed an imaging/radiotherapy trial

in RCC patients with MoAb A6H, an MoAb that

recognizes a kidney differentiation antigen.30,31 Posi­

tive images were obtained in 5 of 15 patients

examined. The low number of positive images was

attributed to the presence of circulating antigen,

and the formation of antigen-antibody complexes.

The number of positive images increased with an

altered dosing schedule, but the number of imaged

lesions remained unsatisfactory.

Based on our targeting studies in RCC-bearing

mice and in ex vivo perfused tumor-bearing kid­

neys, a phase I study with 13iI-labeled MoAb G250

was performed.32 In this protein dose escalation

study, the primary study objectives were evaluation

of the toxicity, pharmacokinetics, and localization

capabilities of 1311 MoAb G250. More than 90% of

primary and metastatic disease as demonstrated by

magnetic resonance imaging (MRI) and computed

tomography (CT) scans was imaged by ml MoAb

G250. Metastatic lesions in lymph nodes, bone, and

lung were visualized. Furthermore, additional meta­

static disease documented at surgery but not de­

tected by MRI and CT scans was visualized. For example, radioimmunoscintigraphy with l3lI MoAb

G250 showed a small hot spot in the liver of one of

the patients which was not visualized by other

means. Nine months later, the patient showed

recurrence of RCC in that precise location. 1311i

MoAb G250 scanning also revealed diffuse meta­

static RCC not recognizable by MRI or CT in a

polycystic kidney of another patient.

From the ability of MoAb G250 to yield sharp

images and visualize small tumor lesions it was

MoAb G250 IN RENAL CANCER 37

concluded that MoAb G250 has considerable poten-

tial as an imaging agent. However, not all primary

or metastatic RCC lesions express G250 antigen,

and some express it only in a minority of cells. This

emphasizes the need for additional RCC-specific

MoAbs because problems of antigen heterogeneity

have to be overcome for therapeutic efficacy.

The absorbed dose to the tumor delivered by the

best MoAb/radionuclide combination studies thus

far has been calculated at 2,000 to 3,000 cGy, while

maintaining less than grade IV toxic effects.27

Vaughan et al calculated the minimal requirement

for effective therapy with 13lI and 90Y-labeled

MoAbs injected intravenously assuming an avarage

accumulation of 0.005% of the administered dose/g

of tumor and a maximum reasonable whole body

dose of 2 Gy.33,34 These investigators concluded

that the tumor uptake should be increased by a

factor 10 for effective therapy, Calculation of the

maximal fraction of the injected dose 1311 MoAb

G250 recovered in tumor sites showed that this was

generally 10 to 100 times greater than previously

reported accumulation of radiolabeled MoAb in

solid tumors. The mean accumulation of the admin­

istered dose was also approximately tenfold higher

than previously reported, fulfilling die require­

ments of Vaughan et al.33,34

Based on these findings, a phase I/II trial with

escalating doses of 131I labeled to 10 mg of MoAb

G250 was undertaken in groups of three patients

with inoperable metastatic RCC.35 Thus far, 21

patients have been treated. The maximum adminis­

tered dose has been 90 mCi/m2. Targeting of

radioactivity to all known sites of disease was seen

in all G250-antigen positive patients (19 of 21).

Elevation of hepatic enzymes was observed in 18

patients, starting approximately 10 days after treat­

ment and returning to baseline by 3 weeks. This

elevation is probably due to MoAb G250 accumula­

tion in the liver. G250 antigen is expressed in the

larger bile ducts, and examination of liver biopsy

specimens in the phase I protein dose escalation

trial revealed MoAb G250 accumulation in bile

duct epithelium. However, the amount of MoAb

G250 necessary to saturate the hepatic compart­

ment was minimal, and estimated in the range of

200 ]xg. Nevertheless, this amount seems to be

sufficient to induce mild liver toxicity. At the

75-mCi/m2 dose level, one of six patients had

reversible grade IV thrombocytopenia, with a nadir

at 4 weeks. No other major toxicity has been

observed. There have been no major responses, but

it is encouraging that stable disease was noted in 11

patients up to 9 months postradioimmunotherapy,

as these patients with disseminated inoperable

RCC tend to do very poorly.

CHIMERIZED MoAb G250

Generally, administration of murine MoAbs to

patients elicits a human antimouse antibody

(HAMA) response. This HAMA response is often

directed against the Fc part of the immunoglobulin

and independent of the amount of MoAb, or

administration route. In all sera obtained from

MoAb G250-injected RCC patients, HAMA of

the IgM and IgG subclasses were detected.32 For

repeated administration, eg, multiple radioimmuno­

therapy or multiple treatment with naked antibody

to induce ADCC, HAMA responses need to be

minimized because circulation HAMA reduces the

tumor uptake of MoAb at subsequent administra­

tion because of MoAb-HAMA cross-linking. With

recombinant technology, mouse MoAb variable regions can be grafted onto human Ig constant

regions, and these constructs can be subsequently

transfected into mammalian cells, which now pro­

duce chimeric immunoglobulin.36'38 Substitution of

the mouse Fc part by human Fc has the additional

advantage that all Fc-related effector functions are

now matching the human effector cells. Principally,

the use of chimeric IgG should augment ADCC

mediated tumor cell lysis, and unmodified chimeric

MoAb might be suitable to destroy (minimal)

residual disease. Chimerized antibodies are ex­

pected to be less immunogenic in man. We have

produced chimeric MoAb G250 (lgGl subclass)

and are currently testing their efficacy in ADCC

and will start a phase I protein dose escalation trial

to investigate the pharmacokinetics and targeting

ability of chimerized MoAb G250.

G250-RELATED ANTI-IDIOTYPE ANTIBODIES

In the preceding sections, the use of MoAb G250

as a passive immunotherapeutic agent was dis­

cussed. Another strategy is to use MoAbs to induce

active immunization. In 1974̂ Jerne proposed that

the immune system is at a steady state by an

equilibrium of lymphocytic clones bearing comple­

mentary receptors.39 This equilibrium can be dis­

turbed either by a foreign or self-antigen, resulting

in a response which aims at the restoration of the

balance. In this response, anti-antibodies (Ab2)

38 OOSTERWIJK, DeBRUYNE, AND SCHALKEN

play a major role. This hypothesis has become

known as the network theory and has been substan­

tiated by many investigators. The critical feature of

this theory is that the binding pocket of the

antibody that determines its specificity elicits anti-

antibodies. Several so-called idiotypic determinants

can be present on one immunoglobulin molecule.

In some cases anti-idiotypic antibodies will mimic

the original antigen. These so-called internal image

Ab2 offer promise as “vaccines” to various infec­

tious agents, and they might be used to induce

immune responses to tumor antigens.40'46 One of the advantages of this appealing approach is the

long-lasting protection that vaccination with TAA- mimicking MoAbs might achieve. Principally, the

requirements of these internal image MoAbs are

the same as those for MoAbs recognizing TAA that

might be useful for passive immunotherapy. Cross­

reactivity with normal tissues should be minimal, to

minimize undesired side effects related to organ

toxicity elicited by vaccination with internal image

MoAbs resembling normal differentiation antigens.

Chattopadhyay et al have demonstrated that vacci­

nation with Ab2 resembling a melanoma-associ­

ated TAA that is expressed on a restricted number

of normal tissues can induce TAA-specific re­

sponses, indicating that TAA expression on a

restricted number of tissues does not hamper the

successful implementation of Ab2 vaccination.47

Considering the restricted expression of G250,

vaccination with internal image Ab2 resembling

this RCC-T A A might represent a new therapy for

RCC patients. Therefore, we have started to isolate

Ab2 bearing the internal image of MoAb G250. Six

MoAbs were isolated after fusion of spleen cells

from mice immunized with MoAb G250 which

showed a dose-dependent inhibition of binding of

i25I-MoAb G250 to antigen. Because Abl-Ab2

interactions in close vicinity of Ab 1-antigen inter­

actions will interfere with Ab 1-antigen binding,

competition for antigen binding was insufficient

evidence to conclude that true internal image Ab2

were isolated. To investigate whether these Ab2

could substitute for the initial G250 antigen, mice

and rabbits were immunized with the Ab2 to

monitor for the occurrence of Ab3 resembling

MoAb G250 (so-called Abl'). Indeed, for all six

Ab2 Abl' antibodies were detected, showing the

functional identity of the six MoAbs and G250

antigen.48

Initially, these six MoAbs were divided into two

immunologically distinct groups, recognizing two

partly overlapping epitopes.48 However, additional

fine specificity studies have shown that the six

internal-image MoAbs can be divided into four

mutually exclusive groups, evidence that the six

Ab2s recognize four slightly different idiotopes in

the Abl binding pocket.49 Because they seem to

recognize unique epitopes in the MoAb G250

binding pocket, it implies that not all six internal

image MoAbs are true representations of G250

antigen. Consequently, one of the MoAbs may

show superior antitumor characteristics.

To investigate the antitumor efficacy of immuni­

zation with Ab2, serum of Ab2-immunized mice

was transferred to nu/nu BALB/c mice challenged

with human RCC cells. In this Winn-type assay,

transfer of any Ab3-serum resulted in significant

tumor growth inhibition (4 weeks; P < .05 versus

all control groups, 8 weeks; P < .01 versus all

control groups) and lower tumor take as compared

with control groups (Table 1). Four weeks after

tumor challenge, no significant difference between

Ab3 treatment groups was seen. Small tumors were

detectable in all Ab3-treated animals. However, 8

weeks after tumor challenge (3 weeks after cessa­

tion of therapy), remarkable differences between

the different treatment groups were observed (Fig

1). All mice treated with Ab3-91 showed complete

tumor regression. Macroscopically as well as micro­

scopically no tumor remnants could be detected.

Ab3-31 treatment resulted in tumor regression in

the majority of animals and tumor stabilization of

the remaining tumors. Similarly, Ab3-82 treatment

effected tumor regression in the majority of ani­

mals, with extremely slow growing tumors in the

remaining animals. After Ab3-71 treatment, slow

growing tumors were measurable in all animals.

The doubling time of tumors remaining after Ab3

treatment was significantly lengthened. Four weeks

after cessation of therapy the tumors started to

grow with doubling times comparable to control

tumors. It is likely that 4 weeks after discontinua­

tion of therapy the circulating levels of Abl'

antibody and serum factors were depleted and too

low for continuation of tumor control. Likewise,

the early growth of the tumors treated with Ab3-91

can be explained by a dose effect. Because the Abl '

titers were moderate, several treatments may be

necessary to achieve sufficient high levels of Abl'

for tumor control and regression. Based on these

results we conclude that NUH-91 immunization

MoAb G250 IN RENAL CANCER 39

Table 1. Summary of Representative Protection Experiment With Ab3 Sera•

TreatmentNo. of Mice

Tumor Take (% ) 4 w 8 w

Tumor Volume (mm3 ± SE) 4 w 8 w

Ab3-31 5 100 80 9.8 ± 2.5* 11.8 ±5.6+Ab3-71 5 100 100 10 ± 2.2* 26.7 ± 5.2+Ab3-82 6 100 66.7 8.1 ± 2.7* 17.9 i 8.7+Ab3-91 6 100 0 7.7 ± 2.2* 0+Nontreatment 6 100 100 23.5 ± 4.4 126 ± 16.6Ab3-MOPC 5 100 100 29 ± 7.5 98.9 ± 12.3NMS* 5 100 100 31.8 ± 14.4 114 ± 21.0

*P < .05.tP < .01 compared with last three cases in this list. ^Abbreviation: NMS, treated with normai mouse serum.

elicits the most powerful antitumor effects, and

that this Ab2 might be the best candidate for

clinical trials.

Because the animals were treated with whole

serum, the antitumor effects can also be explained

by circulating serum factors, eg, T cell factors and

cytokines. We and others have shown that combi­

nation therapy with BRMs, which are serum circu­

lating factors, can result in significant tumor growth inhibition. Treatment of established RCC xeno­

grafts with interferon-alfa, tumor necrosis factor

and MoAb G250 resulted in tumor growth inhibi­

tion and tumor regression.23 However, preliminary

experiments have shown that Ab3 treatment is

superior to BRM/MoAbG250 treatment. It is there­

fore likely that at least part of the antitumor effects

are Abl' related. We are currently investigating

the therapeutic efficacy of Ab3 serum in mice with

established RCC xenografts.

The tumor regression induced by Ab3 serum

transfer could also be explained by the presence of

T cell factors such as specific macrophage arming

factor (SMAF) and mast cell T-Cell factor (MTCF).

These factors can, among others, provide antigen-

specificity to nonspecific cells.50'52 Immunization of

immune competent mice with any of the six Ab2

investigated results in profound transferable anti­

gen-specific cell-mediated immunity, with concomi­

tant serum transferable early delayed-type hyper­

sensitivity responses, attributable toT cell factors.53

Because these factors persist approximately 2 to 16

days after immunization,50 and animals were treated

with immune serum harvested 7 days after immuni­

zation, part of the antitumor effects may have been

C Aba -31

Fig I. Individual tumor growth curves of RCC challenged mice treated with Ab3 sera. BALB/c nu/nu mice received s.c. injections of 10s SK-RC-52 cells (G250-posi- tive human RCC cell line) in the right flank, and treatment was started. Four groups of mice were treated with i.p. injections of NUH» Ab3 sera, and three groups served as controls: no treatment was given (None), mice were treated with serum from MOPC21-KLH immu­nized mice, or mice received nor­mal mouse serum (not shown). Tu­mor volumes are expressed as mean ± SE. Figure is representa­tive for several independently per­formed experiments.

200-aaaaa|

150-cm m

3 100 -

050-

< 0 ■?0 3

c3 200-

0150-

m m

3“ 5

100-

50-

n J

D Aba -71200 -

150-

100 -

E Aba -82200 -

150-

100 -

50-

F Ab3-91

3 4 5 6 7 8

week

40 OOSTERWIJK, DeBRUYNE, AND SCHALKEN

elicited by T cell factors. It is likely that similar

factors, with similar anti tumor efficacy will be

induced by Ab2 immunization of RCC patients.

FUTURE PROSPECTS

Our future efforts will be directed at investigat­

ing the targeting ability of chimerized MoAb G250.

We anticipate that chimerization of murine MoAb

G250 will allow multiple treatments, because hu-

man antimouse responses are reduced. If that is

true, multiple treatments with, eg, high-dose l31I-

labeled chimeric MoAb G250 would become fea-

sible. Once the targeting ability and pharmacokinet­

ics of chimeric MoAb G250 are known, we intend

to investigate the radioimmunotherapeutic poten­

tial of high-dose 131I-labeled chimeric MoAb G250.

In addition, chimerization may provide the anti­

body with appropriate effector functions. This will

enable engagement of human effector cells with

chimeric MoAb-G250 coated tumor cells, effectuât-

ing tumor cell lysis.

We will also pursue the possibilities of anti­

idiotype therapy. Much needs to be resolved with

respect to the identification of the mechanism

behind the observed antitumor efficacy. Currently

we are in the process of identifying which effector

cells are involved in tumor cell elimination, and are investigating which factors are of prime importance

for tumor regression. Regardless of the effector

mechanism, our observation that Ab2 vaccination

results in tumor regression is of clinical relevance

because it seems to indicate that tumor immunity

can be acquired. Patients at risk for tumor recur­

rence, eg, patients with T3NoMo disease, may

benefit from adjuvant treatment by Ab2 vaccina­tion.

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