Induction of Antitumor Immunity by an Anti-Idiotype Antibody Mimicking Carcinoembryonic Antigen Shehla Pervin, Mala Chakraborty, Malaya Bhattacharya-Chatterjee, et al. Cancer Res 1997;57:728-734.
Updated version
E-mail alerts Reprints and Subscriptions Permissions
Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/57/4/728
Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at
[email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at
[email protected].
Downloaded from cancerres.aacrjournals.org on April 23, 2014. © 1997 American Association for Cancer Research.
[CANCERRESEARCH 57.728-734,February15, 19971
Induction of Antitumor Immunity by an Anti-Idiotype Antibody Mimicking Carcinoembryonic Antigen Shehia
Pervin,
Mala Chakraborty,
Malaya
Bhattacharya-Chatterjee,
Hasan
Zeytin,
Kenneth
A. Foon, and
Sunil K. Chatterjee' Department of Obstetrics and Gynecology [S. P, S. K. C], Department of internal Medicine, Division of Hematology and Oncology [M. C., M. B-C., K. A. F.], Department of Microbiology and immunology [H. Z], and The Lucille Parker Markey Cancer Center, [S. P., M. C., M. B-C., H. 1, K. A. F., S. K. C.], University of Kentucky Medical Center, Lexington, Kentucky 40536.0096
ABSTRACT Carcinoembryonic
antigen
(CEA) is a tumor-associated
antigen
ex
pressed on most gastrointestinal adenocarcinomas and is a putative target for cancer immunotherapy. We developed a murine monoclonal anti idiotype (anti-Id) antibody, 3H1, which mimics a specific epitope of CEA, for cancer immunotherapy. In this study, the efficacy of 3H1 as a tumor vaccine was evaluated In a murine tumor modeLIn this model, the murine colorectal
cancer cell line MC-38 was transduced
with the human
CEA
gene and injected into syngeneic C57BL/6 (H-2―) mice. Immunization of naive mice with 3H1 conjugated with keyhole limpet hemocyanin Freund's adjuvant induced humoral and cellular anti-3H1 as well as anti-CEA immunity. Mice Immunized with 3H1 were protected against a challenge with lethal doses of MC-38-cea, whereas no protection was observed when 3H1 vaccinated mice were challenged with CEA negative MC-38 cells or when mice were vaccinated with an unrelated anti-Id
evoke a very poor immuneresponse in a tumor-bearinghost due to a state of T cell-mediated suppression(4, 5) as well as to the develop
antibody and challenged with MC-38-cea cells (P < 0.003). These data demonstrate that the 3H1 vaccine can induce protective CEA-specific antitumor
ment of antigen tolerance (6). An internal image antigen expressed in a different molecular environment may overcome immunosuppression in the host by stimulating “silentclones―and/or by allowing T-cell help to become active, making the overall immune response stronger
immunity.
INTRODUCTION Active specific immunotherapy is an attractive approach to cancer therapy in the adjuvant setting. This therapy is intended to boost the host's antitumor immune response, in contrast to passive immuno therapy, which requires the infusion of large doses of antibodies or cells. Classic active immunotherapy is based on immunization with TAAs2 extracted from tumors or TAA obtained by recombinant DNA technology.
There
based on the region of the variable domain of the Abi they recognize (2). Ab2a recognizes idiotopes that are outside the antigen binding site. Ab2f3 recognizes the binding site of Abl and resembles the original epitope recognized by Abl. If the target idiotope is close to the binding site so that it can interfere with antigen binding, it is called Ab2-y (2). Because Ab2f3 represents essentially the internal image of the antigen, this idiotopic antibody can be used as a surrogate TAA. This cascade of complementary idiotopes is the basis of making idiotype vaccines for cancer as well as for bacterial, viral, or parasitic infections (reviewed in Ref. 3). Our goal is to use this immunother apeutic approach for cancer patients. There are several advantages for the use of a surrogate TAA such as an Ab2f3 for cancer immunotherapy. Ab2j3 is more explicitly “foreign― than is TAA. The TAAs are often part of “self' and usually
are several
problems
with the use of TAA
from
tumors: (a) it is difficult to obtain sufficient quantities of purified TAAs for immunization; (b) TAAs are often chemically ill defined and not easily reproduced; and (c) use of TAAs as an antigen has the potential for transmitting putative contaminating tumor viruses. Al though this difficulty can be circumvented by the use of TAAs prepared by recombinant technology, there are insufficient data from trials with recombinant TAAs to fully assess the potential of this approach. One area of active immunotherapy involves the use of anti-idiotype antibodies. This idea is based on Jeme's network concept (1), which can be summarized as follows. A given antibody (Abi) reacts with epitopic determinants on an antigen. Although antigenic determinants are mostly found on foreign macromolecules, structural determinants on the variable regions of an Abl can also serve as determinants that are recognized by a second antibody (Ab2). The “epitopes― on an Abl that are recognized by an Ab2 are called idiotopes, and the Ab2 is an anti-idiotopic antibody. The Ab2 can be classified into three types
(7).Furthermore,the immunologicalstatusof a cancerpatientis often suppressedand only able to respondto certainT-dependentantigens and not to other antigen forms. We have generated a murine monoclonal Ab2(3 designated 3H1 (8), which mimics a distinct epitope of the TAA, CEA. CEA is present at high density on tumor cells of more than 95% of colorectal, 70% of lung adenocarcinoma, and 50% of breast cancer patients (9—1 1). We have shown previously that 3H1 can induce anti-CEA antibody in small animals (8) and nonhuman primates (12). In a Phase I clinical trial of advanced colorectal cancer patients, we further demonstrated that 3H1 can induce anti-CEA immunity in humans (13). In these studies, although induction of anti-CEA immunity by the surrogate antigen could be demonstrated, a definitive antitumor effect of the induced immunity could not be shown. This was in part due to the fact that suitable animal models were not available for the precinical studies, and the cancer patients included for the PhaseI study were all, of necessity, advanced stage patients with large tumor burdens and limited life expectancy. In this report, we have used a murine model to demonstrate that immunity induced by multiple injections of the Ab2@, 3Hl, can protect mice against a challenge by lethal doses of CEA-positive tumor cells.
MATERIALS Materials.
Received8/5/96;acceptedI 2/18/96. 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. @
Gynecology,204CombsResearchBuilding, Universityof KentuckyMedicalCenter,800 Rose Street, Lexington, KY 40536-0096. abbreviations
used are: TAA,
tumor-associated
antigen;
CEA,
METHODS
Purified CEA was obtained commercially
from Rougier Bio
tech (Montreal,Quebec,Canada).This materialwas isolatedfrom a human liver metastasis of colonic adenocarcinoma by perchloric acid extraction and
To whom requestsfor reprintsshouldbe addressed,at Departmentof Obstetricsand
2 The
AND
carcinoembryonic
antigen; KLH, keyhole limpet hemocyanin; ADCC, antibody-dependent cellular cytotox
icily.
was purified twice by ion-exchange chromatography followed by gel filtration and high-performance liquid chromatography. Purity of this preparation was checked by high-performance liquid chromatography, SDS-PAGE, and immu
noprecipitation(8). The monoclonalanti-CEA antibody, 8019 (IgGlK) has been described previously (8). MC-38 murine colon adenocarcinoma (14) and human CEA transduced murine carcinoma cells, MC-38-cea (15), were kindly
728
Downloaded from cancerres.aacrjournals.org on April 23, 2014. © 1997 American Association for Cancer Research.
AN'IlTUMOR IMMUNfl'Y BY ANTI-IDIOTYPE ANTIBODY
provided by Dr. Jeffrey Schlom (National Cancer Institute, NIH). Female C57BLi6 mice (6—8weeks of age) were obtained from Harlan Bioproducts for Science, Inc. (Indianapolis, IN). Cell culture media were from Life Technol
added and mixed gently with the stimulants (500 ng/well).
ogies,Inc. [H3]Thymidine,[‘251]KI and[51-Cr]sodiumchromatewereobtained from DuPontNEN. Vaccine. Procedures for the purification of anti-idiotype antibody 3Hl
was calculated from samples with a SD less than 10%. Stimulation indices
have been described (8). Two control anti-idiotype antibodies were also used
in this study.Oneof them,llDlO (16), mimicksanepitoperelatedto a breast cancer-associated antigen, human milk fat globule, whereas 1A7 (17) mimics a melanoma-associated antigen, ganglioside0D2@Anti-idiotype antibodies were conjugated to KLH by glutaraldehyde, and the conjugates were dialyzed extensivelyagainstPBS(18). Immunization.
@
The first immunization
with anti-idiotype conjugates was
performed i.p. after emulsification with Freund's complete adjuvant. The dose of immunogenper mousewasadjustedto contain50 @g of immunoglobulin protein per 100 @.d of PBS. Subsequentimmunizationswere performeds.c., usingthe samedose,after emulsificationwith Freund'sincompleteadjuvant. Scm were drawnfromthe tail vein beforeeach immunizationandwere stored at —20°C. Radioimmunoassay
for
Detection
of Anti-3H1
Antibodies
After 4 days of
incubation in a tissue culture incubator, the cells were pulse chased with 1 @Ci [H3]thymidine.Eachassaywasperformedat leastin triplicate, andthe mean were obtainedby dividingthe meancpm of each sampleby the cpm obtained using the medium without the stimulant. ADCC. Targetcells, MC-38-cea, were labeled by incubationof 2 X 106 cells/0.5
ml of serum-free
DMEM
medium
with
200 pCi
of [5tCr]sodium
chromateat 37°C for 1h. After thoroughwashing,thecellsweresuspendedin serum-free DMEM at a concentration of 1 X 10'@cells/25 @.dof medium. Spleen cells were isolated from immunized mice as described above for use as effectorcells. Labeledcells wereaddedin 96-well plates,1 X l0@'cells/well followed by the additionof 100 @l of diluted mouseserum,and 1 X 106of effector cells in 50 @,tl of serum-free DMEM. Plates were incubated at 37°C for 4 h. Aliquots
from
the supernatants
were counted
in a gamma
counter.
Spontaneousreleasewasdeterminedby assaysof radioactivityreleasedfrom 51Cr-labeled cells incubated in DMEM without effector cells. For the deter
mination of total 1Crrelease, 100 @.d of 1% Triton X-lOOwere added to each well. Sera from mice immunized with PBS were used as control. All assays
were performedat least in triplicate,and specific lysis was calculatedby:
(Ab3).
Falcon plates (96-well) were coatedwith 250 ng/well of the anti-idiotype —
antibody overnight at 4°C.After blocking with 1% BSA in PBS for 2 h at room
temperature,the plateswerewashedextensivelywith PBS.Serawerepooled from three mice and diluted with PBS (40—320-fold),and 50 s.d/well were added. After incubation at room temperature for 2 h, the plates were washed with PBS. Fifty p3 of ‘251-labeled antibody (100,000 cpm/well) were added to
% release=
Experimental release
spontaneous release
Total release—spontaneousrelease
x 100
Murine Model for Determining the Efficacy of 3H1 Vaccine. A murine tumor model expressing human CEA was developed by Dr. Jeffrey Schlom at
the NIH. Murine colorectal carcinoma cells, MC-38, were transduced with humanCEA (15).Thetransducedcell line, MC-38-cea.constitutivelyexpress a gammacounter.Assayswereperformedat leastin triplicate, and samples CEA in culture.Dr. Schlomkindly providedus with the CEA-transducedand with a SD less than 10% were used to calculate the mean. nontransduced cell lines. When these cells are injected s.c. (5 X l0@cells/ ELISA for Detection of Anti-CEA Antibodies. ELISA plates (96-well) mouse) into syngeneic C57BL16 (H@2b)mice, tumors develop in 100% of the were coated with 100 @l of a solution of purified CEA, 250 ng/well overnight mice within 10—15days. at 4°C.Wells were blocked with 1% BSA in PBS for 1 h at room temperature. Cell Culture. MC-38 and MC-38-cea were grown in DMEM containing Fifty @lof pooled serum after dilution with PBS (20—160-fold)were mixed 10%FCS, 1%L-glutamine, 100 ,tWmipenicillin,and0.25 j.@g/m1 streptomy with 50 @l of 0.1%Tween-20/2%BSA in PBSandaddedto eachwell. After cm. MC-38-cea were cultured in the presence of 200 @Wml neomycin ana incubation at room temperature for 2 h, the plates were washed with 0.05% logue G-4l8. Cells (50—60%confluent) were harvested from flasks following Tween20/1%BSA in PBS.One hundred @.d of goat anti-mouseIgG conju a brief treatment with trypsin. gatedwith alkalinephosphatase (1:1000-folddilution) wasaddedto eachwell. Survival of Mice Immunized with 3H1 and Control Vaccine. Female The plates were incubated at room temperature for 2 h. After a thorough C57BL/6 mice (6—8weeks of age) were immunized with 3H1-KLH conjugate washing,100,.dof phosphatase substrate(SigmaChemicalCo.) dissolvedin or control vaccines(11D1O-KLH)as describedabove.A shamvaccination diethanolamine buffer (50 mg of substrate per 50 ml of buffer) were added to with PBS was also performed for comparison. Each group of immunized mice each well. The absorbanceat405 nm was determinedin an ELISAreaderafter was further divided into two equal groups for tumor challenge. Tumor chal 30 mm at room temperature.Assayswereperformedat leastin triplicate for lenge was performed with 5 X l0@of CEA-positive MC38-cea or CEA eachsample,andvalueswithin a 10%variancewereincludedfor calculation negative MC-38 cells. Growth oftumor and survival were monitored daily, and tumor volumewasdeterminedweekly. of the mean. Statistical Evaluation. Statistical evaluation was performed using Idiotype Analysis of Ab3. ELISA plates (96-well) were coated with 100 @.d of a solution of 8019 (Abl) or 3H1 in PBS and blocked with BSA as SigmaStat software (Jandel, San Rafael, CA). P < 0.05 was considered to described above. To each well, 50 @d ofdiluted serum and 50 @l of ‘251-labeled indicate statistical significance. each well, and the incubation was continued at room temperature for another 2 h. After washing with PBS, counts remaining in the wells were measured in
antibodies (3H1 or 8019, as appropriate) containing 100,000 cpm in 1% BSA
in PBS were added simultaneously. After incubation at room temperature for 2 h, theplateswerewashed,andtheradioactivitywasdeterminedasdescribed RESULTS above.For determiningthe inhibition of bindingof ‘251-labeled 8019to CEA, Development of Anti-Anti-Idiotype Antibody. To induce Ab3 CEA-positive MC-38-cea cells (1 X 105/well) were placed in cell culture production in the sera, we immunized C57BL/6 mice with 3H1-KLH plates(no. 107assayplateswith filter; V.P.Scientific,SanDiego,CA), reacted conjugate. Ab3 produced in the sera of these allogeneic mice was with 50 @&l of dilutedmouseserumplus50 @.d of ‘251-labeled 8019solutionin 1% BSA-PBS, at room temperature for 2 h. The filter from each well was taken estimated by a RIA using serum pooled from three randomly selected and Methods.― Data in Fig. 1A show out for determiningthe radioactivity after thoroughwashing.Pooledserum mice, as described in “Materials from normal mice was used as control in these experiments. that Ab3 is detectable in the sera of immunized mice even after a
300-folddilution.3Hl inducedlowbutdetectable levelsof Ab3in 18
Immune Flow Cytometry. Both CEA-negative MC-38 and CEA-positive MC-38-cea cells (1 X 106 cells/tube) were reacted with 100 @.d of either undiluted mouse serum or 100 ,.tl of PBS or 100 @tl(2 jig) of Abl (8019) for
of 20 mice after two weekly immunizations. However, significant
Ab3 wasgenerated afterimmunization threetimes,andthelevel
1h at4°C. After washing,thecellswereincubatedwith goatantimouseF(ab')2 peaked after 5 weeks of immunization (Fig. 1B). Two controls were IgG-FITC-labeledantibody(Tago,Inc., Burlingame,CA) for 30 mm at 4°C. used in this assay. In the first control (Ab2-Cl), plates were coated The labeled cells were washed twice, fixed in 2% paraformaldehyde, and with anti-idiotype 1A7, which mimicks an epitope on the ganglioside analyzed by immune flow cytometry (FACStar; Becton Dickinson, San Jose, GD2 (17), and the binding of labeled 3H1 was determined after CA). T-Cell Proliferation Assays. Spleen cells pooled from three randomly incubation with 3Hl-immunized mice sera. For the second control, plates were coated with 3H1, but serafrom mice immunized with 1A7 selected mice, which were either immunized with anti-idiotype vaccines or PBS, were isolated.In each well of a 96-well Falconplate,2 X l0@cells were (five immunizations) were used for the 3H1 binding assay. Radioac 729
Downloaded from cancerres.aacrjournals.org on April 23, 2014. © 1997 American Association for Cancer Research.
ANTITUMOR IMMUNITY BY ANTI-IDIOTYPE ANTIBODY
sera from mice after five weekly immunizations with the isotype matched anti-idiotype 1A7 conjugate were used as the control in this assay. ELISA with this control serum after a 1:20 dilution had an absorbance of 0.18 at 405 nm, which was less than the value obtained using the preimmune serum (Fig. 2B). Idiotype Analysis of Ab3. Idiotype of Ab3 induced in mice by
14000 -.—
12000
Pre-Im
—.— 3X —A— 5X —I,-- 6 X
A
—.— 7 x
10000
—.-
Ab2-C1
—.— Ab2-C2
3Hl vaccine wasanalyzed bytheinhibition ofbinding Abi toAb2by
8000
(I) 6000
0
0
4000
2000 @
0 . 0
50
100
150
I
I
200
250
300
3@
Serumdilution
the mouse serum as described in “Materials and Methods.― Results of these experiments are summarized in Table 1. Binding of Abl(8019) to 3H1 was inhibited by the serum from five of five immunized mice, regardless of which of the antibody was used to coat the plates (Table 1), suggesting that Ab3 may recognize the same epitope as Abi and may thus contain Abi' antibodies. Inhibition of binding of 1251 labeled 8019 to CEA was determined by using CEA-positive MC-38cea cell. Once again, serafrom five of five mice inhibited this binding, and the inhibition was significant, even after 80-fold dilution of the serum. These results suggested that the Ab3 induced in mice by the 3H1-KLH conjugate share the same idiotype as Abi.
14000
0.7 I2000
E
B
@
A
0.6
—U—3X \\
0 0.5
—A-5X \
-v-7X \
8000
C,) @
ci@
\4\
C.3. 00
-e 0.2
4000
0
2000.
U) .0
@
\
C@ 0.4 6000
0 0
—.— None
\
10000
0@ 0
1
2
3
4
5
6
7
°‘@
0.0 0
8
Number of weekly immunizations
weekly immunizations.
I
I
I
40
60
80
100
I
I
120
140
160
180
Serumdilution
Fig. 1. Induction of anti-anti-idiotype antibody (Ab3) in mice by immunization with 3Hl-KLH conjugate.Ab3 generationwas assayedby a sandwichRJA with 3H1-coated
plates as described in “Materials and Methods.― Sera from three mice chosen randomly were pooled for dilutionwith PBS. A: for controlAb2-Cl, plates were coated with lA7 before the addition of mouse serum; for Ab2-C2, plates were coated with 3H1, but serum samples were from mice immunized five times with 1A7-KLH conjugate. B, cpm bound/wellby the mouseseradiluted 1:40 with PBSwereplottedagainstthe numberof
I
20
0.7
E Co.6
B
0 @0.5
@
@
tive 3Hl bound by both of these controls were equal to or less than the W0.4 preimmune sera at all dilutions (Fig. 1A). 0 C Development of Abi'. Ab3 antibody induced by Ab23 is poly @03 clonal, and a subgroup of the Ab3 has a similar binding site (paratope) .0@' Ias Abl. Because of this paratope similarity, this type of Ab3 is also 0 called the Abi' to indicate that it might differ in its other idiotopes C@) 02 from Abl. Abl' recognizes the cognate TAA and is the only antibody in the Ab3 population that is likely to have the antitumor property. Abl' was assayed by ELISA as described in “Materials and Meth 0.1 I— I T 0 1 2 3 4 5 6 7 8 ods.―Low and variable levels (A@5 ,@ ofO.2l ±0.01; dilution, 1:20) of Abl' appeared in 6 of 20 mice after two weekly immunizations. Number of weekly immunizations Significant levels of Abl', however, appeared after the third immu nization, and the levels peaked (A405 of 0.56 ±0.06; dilution, Fig.2.Induction ofAbl' antibody inmiceimmunized with3Hl-KLHconjugate. Abl' wasassayedby EUSA with CEA-coated,96-well platesasdescribedin “Materials and 1:20) around the 6th or 7th week in virtually all animals immunized Methods― using the sameserum poolas Fig. 1.A, absorbanceat405 am ofthe seradiluted with the 3Hl vaccine (Fig. 2). Abl' can be detected in the sera of with PBS before or after immunization for 3, 5, and 7 weekly injections; B, absorbance immunized mice, even after a 80—160-folddilution (Fig. 2A). Pooled at 405 am of pooledseradrawn at weekly intervalsafter 1:20dilution with PBS. 730
Downloaded from cancerres.aacrjournals.org on April 23, 2014. © 1997 American Association for Cancer Research.
@
r@ @. ANTITUMOR IMMUNITY BY ANTI-IDIOTYPE ANTIBODY
Table 1 IdiozypeanalysisofAb3 inducedin mice immunizedwith 3H1-KLH conjugate―
Development of Cellular Immunity. To evaluate the induction of cellular immunity, spleen cells from sham-vaccinated mice and those from mice immunized with the 3H1 vaccine were prepared. Spleens from at least three mice selected at random were pooled for use in this
of binding coat/dilution3Hl CEA-positive8019 to coat!3Hl SerumAbl-Ab2inhibitionInhibition bindingcells2087±489±353±74078±885±425±68064±972±714±4 binding8019 assay. T-cell
@
a
for the analysis of Ab3 induced in mice immunized
with 3Hl
vaccine are
described in “Materials and Methods.―Serum from each of the five mice was separately
assayedto obtainthemeanandSE. In theAbl-Ab2 bindinginhibition assays,inhibition by controlnormalserumpool variedbetween 10 and 15%,and inhibitionwas complete with 2.5 i@gof' 8019. In the cell binding inhibition assay, inhibition by normal serum was at the background level.
A
D 3H1
E 8019
C
F
proliferation
was carried
out as described
in “Materials
and Methods―using CEA and 3H1 as stimulants. For positive con trols, 2 ,.ag of phytohemagglutimn were used in these experiments. Mean counts from at least triplicate wells, with SD within 10%, were used for the determination of stimulation indices. Results in Fig. 4 show that significant proliferation takes place using 3Hl as a growth stimulant following week 3 of immunization (>4 times control), but a maximum index is obtained from spleen cells isolated from mice immunized for 6 weeks. Stimulation indices with CEA follow a similar pattern, and after 6 immunizations, the index was more than six times the control. Although the degree of stimulation of spleen cells by CEA varied, the results were qualitatively similar in several independent experiments. Spleen cells from PBS-vaccinated mice showed negligible stimulation with either 3H1 or CEA. The stimula tion index of spleen cells from mice after six immunizations with 3Hl-conjugate, in the presence of 1A7 as the stimulant in the assay, was 4.2 ±1.1, which is significantly (P < 0.001) lower than the value obtained with 3H1 as the stimulant (13.2 ±1.4). ADCC of the Sera from Immunized Mice. To determine whether the Abl' generated by 3H1 immunization is cytolytic for CEA positive tumor cells, such as MC-38-cea, ADCC was determined as described in “Materials and Methods.― Results of the ADCC experi ments are summarized in Fig. 5. Although the extent of specific cell lysis by sera from each individual mouse varied (33 ±7, 1:5 dilution; 13.2 ±5, 1:10 dilution), significant ADCC was observed in five of five mice. Negligible ADCC was observed with sera from mice immunized with PBS (Fig. 5). Protection of 3H1 Immunized Mice against Tumor Challenge. Experiments described above show that after five to six weekly immunizations, anti-3H1 humoral and cellular responsesreached their maximum levels. Anti-CEA immune responses follow a similar pat tern. If the immunity induced is effective and specific for CEA, the
14@—
12—
Fluorescence Intensity Fig. 3. Immunofluorescence analysisof CEA-positiveMC-38-ceaandCEA-negative MC-38 cells after reactionwith mice sera. The assay was describedin “Materials and Methods.― A—C, incubationof MC-38-cea cells: A, scm from mice before and after six immunizationswith 3H1-KLH conjugate;B, PBS and monoclonalanti-CEA antibody
)< V
@
@CEA
3H1
ci@ @8H
8019; C, sera from mice before and after six immunizations with isotype-matched,
unrelatedanti-idiotypeantibody,1A7-KLH. D—F, incubationof MC-38 cells: D, before and after six immunizations with 3H1-KLH conjugate; E, PBS and 8019; F, before and after six immunizations with lAl-KLH.
.
E
I @4-
@
Flow Cytometric Analysis ofAbi'. To further verify the nature of Abi ‘ induced by 3H1 vaccination, flow cytometric analysis of pre immune sera and pooled sera from three mice after six immunizations with the 3H1-KLH conjugate was performed. Results presented in Fig. 3 demonstrate that sera from mice immunized with the 3Hl vaccine can bind to the MC-38-cea cell surface (Fig. 3A) identical to the monoclonal anti-CEA antibody, 8019 (Fig. 3B), whereas no bind ing was observed with preimmune sera or sera of mice immunized with isotype-matched anti-idiotype antibody, lA7 (Fig. 3C). CEA negative MC38 cells did not bind sera from mice immunized with 3H1 vaccine (Fig. 3D), monoclonal anti-CEA antibody 8019 (Fig. 3E), or with sera of mice immunized with lA7 (Fig. 3F).
0-•
5X
@i@'
Numberof weeklyimmunizations Fig. 4. Proliferation of splenocytes of mice immunized weekly with 3H1-KLH in the presence of 3H1 and CEA as stimulants. T-cell proliferation was assayed by determining [3H]thymidine incorporation by splenocytes isolated from PBS vaccinated mice (N) and mice immunized weekly with 3H1-KLH as described in “Materialsand Methods.―Index
for a stimulant was determined by dividing the mean cpm from assays in triplicates by the meancpm obtainedwith media without the stimulant.3-6X, number of weekly immunizations.
731
Downloaded from cancerres.aacrjournals.org on April 23, 2014. © 1997 American Association for Cancer Research.
ANTITUMOR IMMUNITY BY ANTI-IDIOTYPE ANTIBODY
60
I20
50
I00
(I) (1)
>@ 40 -J
80
>
2:
0 @30
0
60
C')
ci)
0@
40
@)20
20
10
0 0
0
Fig. 5. ADCC by serum from immunized mice. Means (bars, SD) of specific lysis by each vaccine group consisting of five mice are shown. Sera were obtained from mice immunized six times with either the 3H1 vaccine or PBS. Specific cell lysis was determined as described in “Materials and Methods― using MC-38-cea as target cells. Sera
10
20
30
40
50
60
Daysaftertumorchallenge
were diluted 1:5 and 1:10 with PBS.
@
CEA-positive tumor should be rejected if implanted after this period. Growth of CEA-negative tumors should remain unaffected by this immunization. Mice immunized by six weekly immunizations with 3H1 vaccine were challenged with lethal doses of CEA-positive MC-38-cea cells or control CEA-negative MC-38 cells. In Fig. 6, results of the survival experiments are shown in a Kaplan-Meier plot. In the experiment described in Fig. 6A, 24 mice were immunized with 3Hl immunogen, as describe in “Materials and Methods,― and divided into two groups of 12 mice each. One group was challenged with MC-38-cea, whereas the second group was challenged with MC-38 cells. MC-38 tumors grew in the latter mice without any sign of regression, and all of the mice died within 22 days. However, in the group of mice challenged with MC-38-cea, the tumor grew for about 10 days after the challenge and became hemorrhagic with signs of tumor rejection; then tumor disappeared within 3 days. In the absence of further treatment with 3H1, however, tumors regrew at the chal lenge site. Although all of the mice challenged with MC-38 died within 22 days after challenge, 11 of 12 mice challenged with MC 38-cea survived more than 50 days (P < 0.001). A group of 10 mice was immunized with the isotype-matched control anti-idiotype antibody, 11D1O. In this vaccine group, five mice were challenged with MC-38 cells, and five were challenged with MC-38-cea cells. The results are shown in Fig. 6B. Although mice challenged with MC-38-cea survived slightly longer than those challenged with MC38 (one remained alive for 50 days), the difference in survival between these two groups was not statistically sig nificant (P > 0.3). . . . . Another group of 11 mice were mock vaccinated with PBS. In this group, six mice were challenged with MC-38-cea cells, and five were injected with MC-38. The survival of these mice is shown in Fig. 6C. Except for one mouse challenged with MC-38-cea, which survived more than 50 days, all remaining mice died within 23 days (P > 0.3). To determine the efficacy of 3Hl vaccine against established tumors, we performed some preliminary experiments. Mice were injected with 5 X l0@ MC-38-cea cells, and anti-idiotype antibody therapy was started 3 days after the tumors were injected. Mice were treated by injection with either the 3Hl-KLH conjugate or the 1lDlO-
2: Cl) 0
10
20
30
40
50
60
Daysaftertumorchallenge 120
ioo
80 60 0
40
20
0 0 Days
10
20 after
30 tumor
40 50 challenne
60
J Fig. 6. Survivalof immunized miceafterchallenge with tumorcells.Micewere
KLH conjugateevery4 daysat thetumor injection sitefor six courses immunized bysixweeklyinjections of:A,3H1-KLH; B,isotype-matched anti-idiotype . .
.
antibody 11D1O conjugated to KLH; and C, PBS as described in “Materials and Meth
of treatment. Initially, tumors developed in both groups at the same rate. On completion of the six courses of treatment, tumors of six of
ods.― Oneweekafterthefinalimmunization, micewereinjecteds.c.with5 X l0@MC-38 or MC-38-cea cells.Tumordevelopment andsurvivalweremonitored daily.
732
Downloaded from cancerres.aacrjournals.org on April 23, 2014. © 1997 American Association for Cancer Research.
ANTITUMOR
IMMUNITY
BY AN'fl-IDIOTYPE
nine mice treated with the 3111vaccine became necrotic and showed signs of regression. In the control group, only one of eight mice showed regression. Although a small number of animals was used in these preliminary experiments, the data are interesting and worth repeating. Experiments are under way with a larger number of ani mals, using different doses of vaccine administered by different routes. DISCUSSION The results described in this communication demonstrate that the anti-idiotype antibody 3111, mimicking human CEA, can induce hu moral and cellular immune responsesin C57BL/6 mice, which inhibit the development of tumors after challenge with lethal doses of syn geneic tumor cells. These therapeutic effects are antigen specific, because tumor development was not prevented when the immunized mice were challenged with the same tumor cells that were CEA negative. A therapeutic effect was not observed in mice vaccinated with an isotype-matched unrelated anti-idiotype antibody. Low levels of Ab3 were detected in 18 of 20 mice after two to three immuniza tions and reached their maximum level following five to six immu nizations. Abi' levels paralleled Ab3. T-cell proliferation with CEA as the stimulant also peaked after six immunizations to a maximum of six times the control. Thus, to obtain maximum cellular and humoral immune responses, five to six weekly immunizations were necessary. For CEA to stimulate splenocytes from 3111-vaccinatedmice, it is necessary that 3111 have amino acid sequence homology to CEA. Comparison of the amino acid sequence of 3111 complementarity determining regions with the amino acid sequenceof CEA identified several regions of homology (19). Among these, a peptide present in the light chain complementarity-determiing region 2 showed very
highhomology toCEA.It isof interest that,whenthispeptide was used as a stimulant in the T-cell proliferation assayof peripheral blood mononuclear cells from colorectal cancer patients undergoing 3111 therapy, a significant proliferation was observed in peripheral blood mononuclear cells from patients after 3111 therapy compared to before therapy (19). Ab3 induced in mice by 3H1-KLH vaccine inhibited the Abl-Ab2 binding in this system (Table 1). Binding of Abl to CEA-positive MC-38-cea cells was also significantly inhibited (Table 1). These results suggested the induction of Abl'-like antibodies in the immu nized mice. To further confirm whether the induced Abi' can bind to CEA expressed on the tumor cell surface, we analyzed the sera from immunized mice by immune flow cytometric analysis using both MC-38 andMC-38-cea cells.Theseresultsconfirmedthattherelevant Abi' had been induced by 3111 immunization. We have shown previously that in small animals and nonhuman primates, the 3Hl vaccine can induce Abi' (8, 12). The induction of cellular immunity by 3111in mice was not explored previously. In advanced colorectal cancer patients, alum-precipitated 3111 induced Abl' in 9 of 12 patients and CEA-specific T-cell proliferation in 4 patients (13). Although 3H1 induced human antimouse antibody in these patients, I induction
was
unaffected
in
these
patients.
This
Phase
I
ANTIBODY
For several reasons,we consider CEA an excellent target for active immunotherapy with anti-idiotype antibody. CEA is one of the most well-characterized TAAs. It is a Mr 180,000 glycoprotein, its gene has been cloned and sequenced (20—22).CEA is expressed at high density on the surface of a vast majority of human colorectal carcinomas, gastric and pancreatic tumors, and also on other adenocarcinomas such as breast and lung cancer (9—11). Trace amounts of CEA are present in some normal colonic epithelial cells, whereas it is highly expressed in the fetal gut tissues (23, 24). CEA has been shown to function as a homotypic intercellular adhesion molecule (25). It has been speculated that alterations in its expression in carcinoma cells may lead to a general derangement in cell adhesion and consequent disruption of normal cell-cell interactions, resulting in metastasis. This speculation is supported by results of studies in murine models as well as in cancer patients. For example, the levels of CEA produced by human colorectal cancer cell lines directly correlated with their ability to form hepatic metastasisin nude mice following intrasplenic injection (26, 27). iv. injection of CEA in nude mice prior to intras plenic injection of tumor cells has also been shown to enhance metastases from weakly metastatic colorectal cancer cell lines (28). Results of studies in cancer patients showed that the prognosis of colorectal cancer patients were generally poor following surgery, when their serum CEA levels were high (29). Moreover, tumor cells isolated from patients with high serum CEA levels develop more frequent hepatic metastases in nude mice than those from patients with low serum CEA (30). Recently, it has been demonstrated that human colorectal carcinoma cell lines transfected with cDNA encod ing CEA showed significant increase in liver metastases following intrasplenic injection into nude mice, which could be inhibited by anti-CEA antibodies (31). Our model is not metastatic; therefore, we could not demonstrate any effect of 3111 in cancer metastasis. How ever, if anti-CEA antibodies can inhibit tumor metastases (31), anti CEA antibodies induced by 3111 immunization in colorectal cancer patients (13) are likely to be beneficial to these patients. Several monoclonal antibodies recognizing TAAs have shown to mediate ADCC (32—35);none of them were against CEA. Recently, one such antibody (R4) was made against CEA (36). Significant ADCC was invoked in five of five mice by immunization with a 3H1-KLH conjugate. ADCC could be an additional important mech anism for tumor protection by 3111 immunization. We are currently planning to confirm this observation by adoptive transfer of immune serum into naive mice. Induction of cellular immunity in cancer patients by anti-Id vacci nation has been reported by several laboratories (13, 37—41).Using another Ab2f3, a significant correlation was reported between Ab2(3dependent T-cell stimulation and tumor regression in colorectal can cer patients (41). Taken together, these data show that an Ab2(3 can induce effective T-cell responses. We are at present determining the subtypes of T cells that were induced to proliferate by CEA, the nature of the cytokines released, as well as the roles of various cytotoxic T cells that may be involved in the eradication of CEA-positive tumors in this model.
trial,
however, was designed to determine the safety of the vaccine and examine the induction of immunity in advanced colorectal cancer patients. There was no toxicity in 3111-immunized patients, although CEA was a “self'antigen for them. The antitumor efficacy was not tested in this advanced group of patients but is now being tested in patients in an adjuvant setting with minimum tumor burden. Although these T-cell results in mice may not be directly extrapolated to humans, these results are encouraging and suggest that immunization with an Ab23 has the potential for cancer therapy, particularly in the minimum tumor burden adjuvant setting.
ACKNOWLEDGMENTS We thank Dr. Jeffrey Schlom (NIH) for the MC-38 and MC-38-cea cells,
Dr. L. Kelly (TheLucille ParkerMarkeyCancerCenter)for critical reviewof the manuscript,andIman Nazhatfor technicalassistance. REFERENCES 1. Jerne, N. K. Towards a network theory of the immune system. Ann. Immunol. (Inst. Pasteur), 125C: 373—389,1974. 2. Bomi. C. A., and Kohler, H. Antiidiotypic antibodiesand internal images in mono
733
Downloaded from cancerres.aacrjournals.org on April 23, 2014. © 1997 American Association for Cancer Research.
ANTITIJMOR IMMUNITY BY ANTI-IDIOTYPE ANTIBODY
Isolation and characterizationof full-length functional cDNA clones for human carcinoembryonicantigen.Mol. Cell. Biol., 7: 3222—3230, 1987. 23. Egan, M. L., Pritchard, D. G., Todd, C. W., and Go, V. L. W. Isolation and immunochemicaland chemical characterizationof carcinoembryonicantigen-like substancesin colon lavagesof healthy individuals. CancerRes.,37: 2638—2643, 1977. 24. Fritsche, R., and Mach, J-P. Isolation and characterization of carcinoembryonic antigen (CEA) extracted from normal human colon mucosa. Immunochemistry, 14: 119—127, 1977. 25. Benchimol, S., Fuks, A., Jothy, S., Beauchemin, N., Shirota, K., and Stanners, C. P. Carcinoembryonicantigen, a human tumor marker,functions as an intercellular adhesion molecule. Cell, 57: 327—334,1989.
clonal andanti-idiotypic antibodies.In: J. C. Venter,C. M. Fraser,andJ. Linstrom (eds.), Monoclonal and Anti-Idiotypic Antibodies: Probes for Receptor Structure and Function, pp. 141—149. New York: Alan R. Liss, 1984.
3. Sacks,D. L., Kirchhoff,L. V., Hieny,S., andSher,A. Molecularmimicryof a carbohydrate epitope on a major surface glycoprotein of Trypanosoma cruzi by using anti-idiotypic antibodies. J. Immunol., 135: 4155—4159, 1985.
neic ADJ-PC-5 plasmacytoma inBALB-c mice. III. Induction ofspecific Tsuppres
4. Haubeck, H. D., and Kolsch, E. Regulation of immune responses against the synge
sorcells to the BALB/c plasmacytomaADJ-PC-5during early stagesof tumorigen
esis. Immunology, 47: 503—510,1982. 5. Howie, S. M., and McBride, W. H. Tumor specific T helper activity can be abrogated by two distinct
suppressor
cell mechanisms.
Eur. J. Immunol.,
12: 671—675, 1982.
6. Greene, M. I. Cellular and genetic basis of immune reactivity to tumor cells. Contemp.Top. Mol. Immunol., 11: 81—1 16, 1980. 7. McBride, W. H., and Howie, S. M. Induction of tolerance to a murine fibrosarcoma in two zones of doses—the involvment of suppressor cells. Br. J. Cancer, 53: 707—711, 1986.
26. Thomas,
repertoireof epitopeson carcinoembryonicantigendifferentiallyexpressedin human colon carcinomasversusnormal adult tissues.Cancer Res., 45: 5769—5780,1985. 10. Steward, A. M., Nixon, D., Zamcheck, N., and Aisenberg, A. Carcinoembryonic
11. Vincent, R. G., and Chu, T. M.. Carcinoembryonic antigen in patients with carcinoma
of the lung. J. Thorac.Cardiovasc.Surg.,66: 320—328, 1978. Bhattacharya-Chattezjee,
M. Pre
carcinoembryonicantigen.J. Immunother.,18: 95—103, 1995.
P. H., Fernsten,
Growth potential of human colorectal carcinomas in nude mice: association with the preoperativeserum concentrationof carcinoembryonicantigen.CancerRes., 48: 1689—1692, 1988. 31. Hashino, J., FUkUda,Y., Oikawa, S., Nakazato, H., and Nakanishi, T. Metastatic potential of human colorectal carcinoma SW1222 cells transfected with cDNA antigen. Clin. Exp. Metastasis, 12: 324—328, 1994.
D6i2. Int. J. Cancer, 46: 1021—1028,1990.
33. Herlyn,D.,Herlyn,M.,Steplewski,Z.,andKoprowsky,H.Monoclonalantibodiesin cell-mediatedcytotoxicity againsthumanmelanomaand colorectalcarcinoma.Eur.
J. Immunol.,9: 657—659, 1979.
in vitro and in vivo anti-tumor properties of a T-cell clone generated from murine tumor infiltrating lymphocytes. J. Biol. Response Mod., 9: 499—511,1990. M., Hand,
29. Shively, J. E., and Beatty, J. D. CEA related antigens: molecular biology and clinical significance.Crit. Rev. Oncol. Hematol.,2: 355—399, 1985.
cellcytotoxicityagainsthumancoloncarcinomasenhancedby monoclonalantibody
Chatterjee, M. Immune response to the carcinoembryonic antigen in patients treated with an anti-idiotype antibody vaccine. J. Clin. Invest., 96: 334—342,1995. 14. Fox,B. A., Spiess,P.J.,Kasid,A., Pun,R.,Mule,J.J.,Weber,J. S.,Rosenberg,S.A.
J. A., Salgaller,
P., andJessup,J. M. Carcinoembryonic antigenenhancesmetastaticpotentialof colorectalcarcinoma.Arch.Surg.,125:300—304, 1990.
32. Pendurthi, T. K., Parker, R., Schiom, J., and Primus, J. Lymphokine-activated killer
13. Foon, K. A., Chakraborty, M., John, W. J., Sherratt, A., KOhler, H., and Bhauacharya
P. F., Kantor,
28. Hostetter, R. B., Campbell, D. E., Kerckhoff, S., Cleary, K. R., Ullrich, S., Thomas,
encoding carcinoembryonic
clinical evaluation in non-human primates of an anti-idiotypic antibody that mimicks
15. Robbins,
0. D. The structure,
30. Jessup,J. M.,Giavazzi,R.,Campbell,D.,Cleary,R., Morikawa,K.,andFidler,L J.
antigenin breastcancerpatients:serumlevelsanddiseaseprogress.Cancer(Phila.), 33: 1246—1252, 1974. H., and
J. M., and Steele,
andcarcinoembryonic antigenproduction.Clin.Exp.Metastasis,10: 25—31, 1992.
9. Muraro,R., Wunderlich,D., and Thor,A. Defmitionby monoclonalantibodiesof
K. A., Kdhler,
K. S., Jessup,
Biophys.Acta,1032:177—189, 1990.
Murine monoclonalanti-idiotypeantibodyasa potentialnetworkantigenfor human carcinoembryonicantigen.J. Immunol., 145: 2758—2765, 1990.
M., Foon,
C. A., Saini,
27. Wagner, H. E., Toth, C. A., Steele, G. D., and Thomas, P. Invasive and metastatic potential of human colorectal cancer cell lines: relationship to cellular differentiation
8. Bhauacharya-Chatteijee, M., Mukeijee, S., Biddle, W., Foon, K. A., and Köhler,H.
12. Chakraborty,
P., Toth,
metabolismand function of the carcinoembryonicantigengenefamily. Biochim.
34. Steplewski, Z., Lubeck, M. D., and Koprowsky, H. Human macrophages armed with murine
P. D., and Schlom,
J. Transductionandexpressionof humancarcinoembryonic antigengenein a murine colon carcinomacell line. Cancer Res., 51: 3657—3662, i991. 16. Bhanacharya-Chatterjee,M., Mrozek, E., Mukeçjee,S., Ceriani, R. L., Köhler,H.,
immunoglobulin
G@, antibodies
to tumors
destroy
human
cancer
cells.
Science (WashingtonDC), 221: 865—867,1983. 35. Hellstrom, I., Beaumier, P. L., and Hellstrom, K. E. Antitumor effects of L6, an IgG@ antibodythat reactswith mosthumancarcinomas.Proc.Natl. Acad. Sci. USA, 83:
7059—7063, 1986. and Foon,K. A. Anti-idiotype antibodiesas potentialtherapeuticagentsfor human 36. Bei, R., Moretti, A., Visco, V., DeFilippi, R., Tsang, K., Frati, L., andMuraro, R. Cell breast cancer. in: R. L. Ceriani (ed.) Antigen and Antibody Molecular Engineering in mediated cytotoxicity of human colon carcinoma cells by a monoclonal antibody (R4) BreastCancerDiagnosisandTreatment,pp. 139—148. New York: PlenumPublishing recognizingthecarcinoembryonicantigen(CEA) andCEA-relatedmolecules.Int. J. Corp., 1994. Oncol., 8: 1127—1135, 1996. 17. Bhattacharya-Chatterjee, M., Mrozek, E., Chakraborty, M., Reisfeld, R. A., Köhler, 37. Durrant, L. 0., Buckley, T. J. D,. Denton, 0. W. L., Hardcastle, J. D. Sewell, H. F., H., and Foon, K. A. Syngeneic monoclonalanti-idiotypeantibodiesagainsta mono and Robins, R. A. Enhanced cell-mediated tumor killing in patients immunized with clonal antibodyto humanmelanomaassociatedantigen.J. Immunol., 150: 142A, human monoclonal anti-idiotypic antibody 1O5AD7. Cancer Res., 54: 4837—4840, 1993. 1994. 18. Bhattacharya-Chatterjee,M., Chatterjee, S. K., Valis, S., Seon, B., and Köhler,H. IdiotypevaccinesagainsthumanT cell leukemia.II. Generationandcharacterization 38. Durrant. L. 0., Doran, M., Austin, E. B., and Robins, R. A. Induction of cellular of a monoclonal idiotype cascade(Abl, Ab2, and Ab3). J. Immunol., 141: 1398—
1403,1988. 19. Pervin, S., Shen@att,A., Wang, H-T., Blalock, E. J., Bhattacharya-Chatterjee, M.. Kdhler, H., Foon, K. A., and Chatterjee, S. K. Proliferation of T-cells from colon cancer patients by peptides based on the structure of an anti-idiotype antibody
mimicking CEA. Proc. Am. Assoc. Cancer Res., 37: 473, 1996. 20. Oikawa,
S., Nakazato,
H., and Kosaki,
0.
Primary
structure
of human
immune responses by a murine monoclonal anti-idiotypic antibody recognizing the 79lTgp72 antigen expressed on colorectal, gastric and ovarian human tumors. hit. J.
Cancer, 61: 62—66,1995. 39. Somasundaran, R., Zaloudik, 3., Jacob, L., Benden, A., Sperlagh, M., Hart, E., Marks, G., Kane, M., Mastrangelo,M., and Herlyn. D. Induction of an antigen-specific T and B cell immunityin colon carcinomapatientsby anti-idiotypicantibody.J. Immunol., 155: 3253—3261,1995.
carcinoem
40. Fagerberg,J., Steinnitz,M., Wigzell,H., Askelöf, P., and Mellstedt,H. Human
bryonic antigen (CEA) deduced from cDNA sequence. Biochem. Biophys. Res. Commun.,142:511—518, 1987. 21. Paxton, R. J., Mooser, G., Pande, H., Lee, T. D., and Shively, E. Sequence analysis of carcinoembryonic antigen: identification of glycosylation sites and homology with
anti-idiotypic antibodiesinduceda humoralandcellular immuneresponseagainsta colorectalcarcinoma-associated antigenin patients.Proc.Nail. Acad.Sci. USA, 92: 4773—4777,1995. the immunoglobulin super-genefamily.Proc.Nail.Acad.Sci.USA,84: 920—924, 41. Fagerberg,J.,Hjelm, A-L., Ragnhammar,P.,FrOdin,J-E.,Wigzell, H., andMellstedt, H. Tumor regression in monoclonal antibody-treatedpatients correlates with the 1987. presence ofanti-idiotype-reactive T lymphocytes. Cancer Res., 55: 1824—1827,1995. 22. Beauchemin, N., Benchimol, S., Cournoyer, D., Fuks, A., and Stanners, C. P.
734
Downloaded from cancerres.aacrjournals.org on April 23, 2014. © 1997 American Association for Cancer Research.
