CANCER BIOTHERAPY & RADIOPHARMACEUTICALSVolume 22, Number 5, 2007© Mary Ann Liebert, Inc.DOI: 10.1089/cbr.2006.357

Antitumor Effect of an Antibody AgainstGastrointestinal Cancer–Associated Antigen CA19.9

Zheng Li,1 Xueli Liu,1 Yunxia Wan,2 Wei Wang,3 and Jie Ma1

1State Key Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of MedicalSciences, PUMC, Beijing, China2Group of Adoptive Cell Therapy, Cancer Hospital, Chinese Academy of Medical Sciences, PUMC,Beijing, China3School of Public Health and Family Medicine, Capital University of Medical Sciences, Beijing,China

ABSTRACT

Despite tremendous efforts in developing cancer vaccines and monoclonal antibodies (mAbs), only a fewpassive or active immunotherapeutic modalities have succeeded. The failure may be due to the existenceof immunosuppressive factors, which caused anergy of patients’ immune system. In this study, we dis-covered that CA19.9 is one such immunosuppressive factor, which could inhibit the proliferation of acti-vated human lymphocytes. However, this inhibition could be reversed by a mAb, LC44, which was specif-ically generated against CA19.9. An in vitro cytotoxic experiment showed that mAb LC44 could form animmunocomplex with CA19.9, which subsequently induced the production of cytotoxic T-cells reacting toCA19.9-positive cancer cells. In addition, mAb LC44 could mediate the complement-dependent cytotoxi-city to CA19.9-positive cancer cells. mAb LC44 showed an antitumor effect in immunodeficient mice withcolon cancer burden in the presence or absence of CA19.9. Based on the observations from this study,we postulated that the mAb, LC44, could be a promising antitumor agent for gastrointestinal cancer andworthy of further investigation.

Key words: CA19.9, immunosuppression, antibody treatment, cancer

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INTRODUCTION

A broad spectrum of immune substances exist inthe circulatory system of cancer patients, whichmight be involved in the development of dis-eases.1 Golda et al. examined circulating immunecomplexes (CICs) isolated from the sera of pa-tients with prostate tumor. They postulated that

the composition of the antigen in CICs is relatedto the level of cancer progression.2 In clinicalpractice, the presence of tumor-associated anti-gens (TAAs) in serum is often considered to becorrelated with a poor prognosis in cancer pa-tients, although they are, theoretically, able to in-duce immune responses against cancer. Sincemost tumor antigens are identified as self-ratherthan tumor specific, the weak autoimmunity in-duced by which is usually not visible in cancerpatients because of the regulation of idiotypicnetwork. Apart from this, the poor quality of im-mune responses to tumor antigens in patients canbe attributed to several factors that can vary with

Address reprint requests to: Jie Ma; State Key Laboratoryof Molecular Oncology, Cancer Institute, Chinese Academyof Medical Sciences; PUMC, 100021, Panjiayuan, Beijing,China; Tel.: 86-10-87788885; Fax: 86-10-87788451E-mail: jiema2000@yahoo.com

the characteristics of the tumor antigens. Amongidentified tumor antigens, some are differentiallyexpressed self proteins, to which high-affinity T-cells have been eliminated during early negativeselection, and some other antigens are highly gly-cosylated, that is, mucins, which are stranded inthe endosomes that cannot be processed by anti-gen presenting cells (APCs) and subsequently areunable to induce immune responses.3 In addition,some tumor-associated glycoproteins influencethe differentiation of dendritic cells, leading tothe anergy of T-cells.4,5 Thus, most mucin anti-gens with high glycosylation are known to be im-munosuppressive.

The gastrointestinal carcinoma-associated anti-gen, CA 19.9, which is expressed by more than60% of colorectal carcinoma tissues derived fromvarious patients, is a mucin TAA.6 To date, noevidence has been reported of the immunosup-pressive effect of CA19.9. But, as a mucin,CA19.9 could potentially bind to lectin-like re-ceptors on the surface of macrophages and lym-phocytes to induce apoptosis and/or anergy ofthese cells.7,8 In a second scenario, the circulat-ing CA19.9, due to its carbohydrate characteris-tics, may not be processed by APC.9 Therefore,high levels of circulating and cell-associatedCA19.9 possess the possibility to cause specificand/or generalized immunosuppression in cancerpatients. When examining the ability of CA19.9to induce antigen-specific T-cell response, wefound that CA19.9 accelerated the death of T-cells in a dose-dependent manner. This led us toinvestigate the immunosuppressive effect ofCA19.9. The proliferative study showed that thepresence of CA19.9 in the culture led to the in-hibition of T-cell proliferation.

In the past three decades, there have been manyreports of monoclonal antibodies (mAbs) recog-nizing tumor antigens.10–12 Although some withrestricted specificity were considered to be po-tential candidates for cancer treatment, few havebeen clinically utilized, which may be due to in-sufficient information about their antitumor func-tion, in addition to positive reactivity against tu-mor cells.

In this study, an anti-CA19.9 antibody, LC44,was generated in order to antagonize the sup-pression induced by CA19.9. This mAb inter-acted with CA19.9 in terms of forming an im-munocomplex, which could be further presentedto APCs and induce a production of cytotoxic T-lymphocytes (CTLs). Furthermore, mAb LC44was extremely effective in mediating a comple-

ment- dependent cytotoxicity (CDC). The appli-cation of mAb LC44 to the treatment of coloncancer in an animal model revealed that immu-nodeficient mice supplemented with human pe-ripheral blood lymphocytes (PBLs) responded tothis antibody either in the presence or absence ofCA19.9. These results implied the potentiality ofmAb LC44 as an immunotherapeutic agent fortreating gastrointestinal cancer.

MATERIALS AND METHODS

Antibody Cell Lines, and Reagents

CA19.9 was purchased from Biodesign (Saco,Maine). mAb LC44 was raised by the polyethyl-ene glycol fusion of splenocytes from BALB/cmice immunized with CA19.9 with NS-1myeloma cells. The specificity of the antibody toCA19.9 was carried out by immunoassays and theisotype of the antibody was determined to be IgG3.Human colon cancer cell lines, SW1116 (CA19.9-positive) and Lo Vo (CA19.9-negative) were ob-tained from American Type Culture Collection(ATCC; Manassas, VA) and used to detect the spe-cific antitumor effect of mAb LC44. The antigenof the YAC-1 cell (human lymphoma cell fromATCC) was prepared by a freeze-thaw method.Serum-free medium, T503, used for T-cell culture,was provided by Kohjin Bio Inc. (Sai-bama,Japan). A CellTitel kit (Promega, Beijing, China)was used for the cell proliferation assay.

Animals

Eight (8)-week-old male SCID mice were pur-chased from the Animal Clone Center, ChineseAcademy of Medical Sciences (CAMS; Beijing,China). All of the mice were kept in a specificpathogen-free animal facility at the Animal Re-search Center, Chinese Academy of Medical Sci-ences (Beijing, China) with free access to foodand water. All animal studies were performedwith the approval from CAMS’ Institutional An-imal Care and Use committees.

T-Cell Proliferation in the Presence ofCA19.9 and/or mAb LC44

Normal human PBLs were isolated from he-parinized venous blood by Ficoll centrifugationand plated in 96-well plates at a density of 1 �105 PBLs/well in T503 medium with or withoutinterleukin (IL)-2 (50 U/mL). The cells were

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pulsed with CA19.9 of different concentrationsat 0, 0.064, 0.32, 1.6, 8, 20, 80, 160, 200, 320,640, 1000, or 5000 U/mL. Human albumin at thesame protein levels was used as the control. Then,the plates were incubated at 37°C for 3 days in5% CO2/air atmosphere. The proliferation wasmeasured using a CellTitel kit following the man-ufacturer’s instructions.

The capability of CA19.9 to suppress the pro-liferative ability of activated T-cells was evalu-ated in a cell proliferation experiment. Normalhuman PBLs were isolated from heparinized ve-nous blood by Ficoll centrifugation and primedby the YAC-1 antigen (2 �g/mL) in T503medium. After a 2-day stimulation, the cellswere washed and plated in 96-well plates at adensity of 1 � 105 PBLs/well in T503 medium.The cells were pulsed with the YAC-1 antigenof different concentrations at 2, 4, or 8 �g/mLwith or without CA19.9 (1000 U/mL). Then, theplates were incubated at 37°C for 3 days in 5%CO2/air atmosphere. The proliferation was mea-sured using a CellTitel kit, according to the man-ufacturer’s instructions.

To investigate the effect of mAb LC44 on thisproliferation, 30 �L of CA19.9 at different con-centrations (500, 1000, 2000, and 4000 U/mL)were added to each well of a 96-well plate, whichcontained approximately 2.5 � 105 PBLs/well.Then, mAb LC44 was added at a final concentra-tion of 50 �g/mL. The mouse-unspecific IgG3 an-tibody was used as the isotype control. The plateswere incubated at 37°C for 3 days, and proliferativeT-cell responses were determined in a standard 3H-thymidine incorporation assay. Briefly, the cells ineach well were pulsed with 1 �Ci 3H-thymidine(Amersham Pharmacia Biopharmacy, Shanghai,China) for the last 18 hours of the culture period.Thymidine uptake was determined by scintillationcounting with a Beckman �-liquid scintillationcounter (LS 5801; Beckman, Beijing, China).

Cytotoxic Experiments

Normal human PBLs were obtained from ahealthy donor by histopaque isolation. The cellswere plated at 106 PBLs/well and incubated withCA19.9 (100 U/mL), mAb LC44 (10 �g/mL),IgG3 (10 �g/mL), CA19.9 � mAb LC44, orCA19.9 � IgG3 at 37°C for 1 week. Then, a 51Crrelease cytotoxic assay was performed to targeteither SW1116 cells (strong expression ofCA19.9) or Lo Vo cells (no CA19.9 expression).The effector-target ratio was 10:1. The released

51Cr was determined by a �-counter (Walagac1470, PE; Perkin Elmer, Waltham, MA). The cy-totoxic index was then calculated, according tothe following formula: percent specific lysis �[(sample release counts per minute [cpm] �spontaneous release cpm)/(maximum releasecpm � spontaneous release cpm)] � 100.

Complement-Dependent CytotoxicityMediated by mAb LC44

Approximately 1 � 104 chromium-labeledSW1116 cells were incubated with mAb LC44 (5�g/mL) or IgG3 (5 �g/mL) at 37°C for 30 min-utes. The cells were then washed twice with T503medium and incubated with such a medium ormedium containing 10 �g/mL of human anti-mouse antibody (HAMA) at 37°C for 30 minutes.Cells with only a HAMA addition were used as acontrol. All plates were washed and fresh humanserum (40%) was added and incubated for 4 hours.The released 51Cr was determined by a �-counter(Walagac 1470, PE). The cytotoxic index was thencalculated according to the following formula: per-cent specific lysis � [(sample release cpm � spon-taneous release cpm)/(maximum release cpm �spontaneous release cpm)] � 100.

Evaluation of the In Vivo Effect of mAb LC44

SCID mice were injected via the tail vein with1 � 107 human PBLs. Blood was collected 7 and14 days after reconstitution to verify the effec-tiveness of the graft. Only mice with human IgGlevel higher than 10 �g/mL were utilized for fur-ther experiments.

There were 30 animals considered to be re-constituted on day 14, according to this criterion.These animals were inoculated with 2.5 � 106

SW1116 cells through an intraperitoneal admin-istration. Three (3) days postinoculation, the micewere randomly divided into six groups for 2 in-travenous immunizations: (i) mAb LC44 (120�g/mouse); (ii) mAb LC44 (120 �g/mouse) �CA19.9 (150 U/mouse); (iii) CA19.9 (150U/mouse); (iv) mouse IgG3 (120 �g/mouse); (v)mouse IgG3 � CA19.9; and (vi) phosphate-buffered saline (PBS). The immunization inter-val was 1 week. Mice were monitored daily andeuthanized when they developed cachexia.

Statistical Analysis

The p-value of the animal study was calculated us-ing software DAS 1.0 (Drug and Statistics for Win-

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dows, Anhui Provincial Center for Drug ClinicalEvaluation, Hefei, China). Differences were con-sidered significant only when the p-value of thecomparison was less than 0.05. Survival data wereanalyzed using the method of Kaplan and Meier.

RESULTS

Antibody LC44 Could Reverse InhibitionEffect of CA19.9 on T-Cell Proliferation

To evaluate the immunosuppressive activity ofCA19.9, the proliferative ability of normal hu-man T-cells was detected after 3 days of reactionwith the antigen. As shown in Figure 1, a lowdose of CA19.9 could stimulate the proliferationof T-cells. When the concentration of CA19.9was higher than 8 U/mL, there was a significantreduction in the T-cell proliferation with the ad-dition of CA19.9 in a dose-dependent manner inthe presence or absence of IL-2. Since IL-2 didnot influence the profile of the cell proliferation,we did not add IL-2 in the subsequent experi-ments. In order to further investigate the im-munosupression effect of CA19.9 on activated

T-cells, the antigen derived from YAC-1 cell wasused to prime the T-cells. As plotted in Figure 2,CA19.9 could inhibit the growth of cells activatedby YAC-1 antigen. Of interest, this suppressionwas reversed by the addition of mAb LC44 (Fig.3). This indicated that CA19.9 cross-linking bymAb LC44 might shield some of the epitopes onCA19.9, which caused immunosuppression.When mAb LC44 in a fixed concentration (50�g/mL) was added against increasing concentra-tions of CA19.9, an overall improved prolifera-tive activity was observed, compared with the ir-relevant IgG3 control. When the concentration ofCA19.9 was going higher, the proliferation of T-cells started to decrease. This reflected the pro-cess that when mAb LC44 were saturated uponthe CA19.9 binding, the excessive antigensstarted to be able to abolish cell proliferation. Thedose ratio between antigen and antibody was fol-lowed in the subsequent experiments.

Immunocomplex Could Stimulate CTL Production

As we previously reported, a specific CTL response against ovarian tumor cells can be

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Figure 1. Immunosuppresive activity of CA19.9 on the proliferation of normal human lymphocytes. Normal human lympho-cytes (1 � 105 cells/well) with or without interleukin-2 (50 U/mL) were cultured in the presence of CA19.9 at 0, 0.064, 0.32,1.6, 8, 20, 80, 160, 200, 320, 640, 1000, or 5000 U/mL. Human albumin, at the same protein concentrations, was used as thecontrol. In 3 days, the proliferation of the cells was measured using a CellTitel kit (Promega, Beijing, China).

generated from PBLs with respect to in vitrostimulation with an antibody.13 In order to de-termine whether the CA19.9-LC44 immuno-complex could trigger antigen-specific cyto-toxicity, a 4-hour 51Cr release assay wasperformed. As depicted in Figure 4, CA19.9-positive tumor cells (SW1116) were lysed byantigen- and/or antibody-primed T-cells, whileCA19.9-negative tumor targets (Lo Vo) werehardly affected. This result indicated that CTLresponse was specific to CA19.9-positive tumorcells and such activity could be determined inan allogeneic system. Among all of the stimu-lators, the immunocomplex was the one withthe highest immunogenecity. After a 1-weekexposure to the LC44 � CA19.9 complex,PBLs exhibited a significantly stronger cyto-toxic ability, compared to those stimulated byCA19.9 or mAb LC44 alone, as well as a con-trol IgG3 or IgG3 � CA19.9 mixture.

mAb LC44 Could Mediate CDC ResponseIn Vitro

Some murine antibodies with the IgG2a/b orIgG3 isotype are capable of mediating CDC andantibody-dependent cell-mediated cytotoxicity(ADCC) in humans. The isotype of mAb LC44is IgG3-kappa, which was extremely effective inmediating complement-dependent cytotoxicity(Fig. 5). This cytotoxicity could be enhanced sig-nificantly with the participation of HAMA, whichindicated that the application of mouse mAbLC44 in humans will enable it to induce a strongCDC response.

Treatment of mAb LC44 Led to a LongerSurvival Time in Human PBL ReconstitutedTumor-Bearing Mice

Prolonged survival time was noted in SCID micetreated with either mAb LC44 or the mAb

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Figure 2. Immunosuppresive activity of CA19.9 on the proliferation of YAC-1-activated human lymphocytes. Normal humanperipheral blood lymphocytes (PBLs) were primed by the YAC-1 antigen (2 �g/mL) for 2 days. The cells were plated in 96-wellplates at a density of 1 � 105 PBLs/well and pulsed with YAC-1 of different concentrations at 2, 4, or 8 �g/mL with or withoutCA19.9 (1000 U/mL). Then, the plates were incubated at 37°C for 3 days in 5% CO2/air atmosphere. The proliferation of thecells was measured using a CellTitel kit (Promega, Beijing, China).

LC44 � CA19.9 immunocomplex, comparedwith other groups (p � 0.05). In the clinic, theCA19.9 level above 37 U/mL was defined to bepositive. In this study, we administered 150 Uof CA19.9 for each mouse, which was equiva-lent to about 50 U/mL. Since the mice were in-oculated with tumor cells, we speculated that thelevel of CA19.9 in the serum was higher than50 U/mL. Therefore, a higher ration betweenmAb LC44 and CA19.9 was performed in thein vivo experiment, compared with the in vitroexperiment. The antitumor effect of mAb LC44might be due to the initiation of CDC response.For the group treated with both antibody andantigen, the CTL production should also be in-cluded, according to the in vitro result. No sig-nificant difference between these two groupswas observed (p � 0.664). Conversely, micethat received CA19.9 alone had very low sur-vival rates, which was even lower compared tothe PBS group (Fig. 6). This might be due to the

speculation that CA19.9 reduces the T-cell pro-liferation. A comparable result was shown inmice treated with CA19.9 � IgG3 (p � 0.284).Twenty (20) days after tumor inoculation, all themice without mAb LC44 treatment developedcachexia and were terminated, while 2–3 micein groups treated with either mAb LC44 or mAbLC44 � CA19.9 remained alive. In 1 week, theremaining mice in these two groups developedcachexia and were out of the experiment conse-quently.

DISCUSSION

Advances in cellular and molecular immunologyin the past two decades have accumulated com-pelling evidence, suggesting that the immune sys-tem plays an important role in the control of ma-lignancy. This has not only been demonstrated inexperimental animals with defined immunologic

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Figure 3. Monoclonal antibody (mAb) LC44 could reverse the immunosuppressive effect of CA19.9 on T-cell proliferation.CA19.9 at different concentrations (500, 1000, 2000, and 4000 U/mL) and mAb LC44 at a final concertration of 50 �g/mL wascultured with normal human peripheral blood lymphocytes (2.5 � 105 cells/well). The mouse-unspecific IgG3 antibody was usedas an isotype control. The cells were incubated for 3 days at 37°C, and proliferative responses were determined in a standard 3H-thymidine incorporation assay.

defects, which exhibit greater susceptibility tospontaneous and induced tumors compared withnormal hosts, but also has been observed in pa-tients clinically. For example, the detection ofantigen-specific CIC in serum from cancer pa-tients indicates the existence of host immune re-sponse against tumor antigens.14 Additionally,the accumulation of immune cells, such as tumor-infiltrating lymphocytes (TILs), could often beobserved at tumor sites, which correlates withimproved prognosis.15,16 Despite the existence of such innate or acquired immune responsesagainst tumor cells, tumor masses survive in mostpatients, indicating that the developing cancer isable to avoid detection, escape, or overwhelm theimmune response. Therefore, strategies based onassisting the surveillance operation of the im-mune system might be harnessed for the therapyof established malignancies.

To achieve specific immune responses againsttumors, immunotherapies are designed to directtumor antigens.17,18 Although cancer patients de-

velop immune responses to many tumor antigens,these responses are typically weak and not ther-apeutic. For colorectal cancer patients, the evi-dence that tumor antigens are able to induce a tu-mor-specific response in the autologous setting isstill scarce and few immunologic studies havebeen performed.19 The main challenge to tumorvaccines, based on molecularly defined tumorantigens, is that many tumor antigens are presentin soluble forms in the circulatory system, whichhave been related to immunotolerance developedin cancer patients.20 For example, patients withpancreatic cancer displaying high levels ofCA19.9 are associated with a very poor progno-sis. In this study, we detected the immunosup-pression effect of this antigen directly through aT-cell proliferation study. The result revealed thatthe proliferative activity of normal human PBLscould be inhibited by CA19.9 in a dose-depen-dent manner. CA19.9 belongs to the mucin fam-ily and is heavily glycosylated. Recently pub-lished literature disclosed that glycosylated

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Figure 4. Cytotoxicity induced by an immunocomplex. Normal human peripheral blood lymphocytes (PBLs) were obtainedfrom a healthy donor by histopaque isolation. The cells were plated at 106 PBLs/well and incubated with CA19.9 (100 U/mL),monoclonal antibody (mAb) LC44 (10 �g/mL), IgG3 (10 �g/mL), CA19.9 � mAb LC44, or CA19.9 � IgG3 at 37°C for 1 week.Then, a 51Cr release cytotoxic assay was carried out to target either SW1116 cells (CA19.9-positive) or Lo Vo cells (CA19.9-negative). The effector-target ratio was 10:1.

antigens were stranded in the endosomes after be-ing captured by APCs, preventing further prote-olysis of the antigen and leading to an unsuc-cessful presentation to T-cells.3 This could besupported by other reports demonstrating that theimmunization of mice with the human MUC1(which shares little homology with mouseMUC1) elicited immune responses only whenunglycosylated protein was used.21 Even thoughthe exact mechanism of this inhibitory effectneeds to be further elucidated, we speculate thatthe existence of CA19.9 might consume a largeamount of mannose receptors on the surface ofAPCs, which presumably influences the antigen-presenting capability of APCs and subsequentlydevastates T-cell proliferation. Our current re-sults imply that patients with a high magnitudeof CA19.9 would have general immunosuppres-sion on cellular immunity and a poor prognosis,which coincides with the clinical role of CA19.9as a poor prognostic indicator.

Injection of mAb LC44, the specific Ab forCA19.9, will inactivate and clear the circulat-

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Figure 6. Survival rates in SCID mice engrafted with SW1116 colon cancer cells. Before the inoculation of tumor cells, SCIDmice were injected via the tail vein with 1 � 107 human peripheral blood lymphocytes. Two (2) weeks after reconstitution, theanimals were inoculated with 2.5 � 106 SW1116 cells through an intraperitoneal administration. In 3 days, the mice were ran-domly divided into six groups for 2 intravenous immunizations: (i) mAb LC44 (120 �g/mouse); (ii) mAb LC44 (120 �g/mouse) �CA19.9 (150 U/mouse); (iii) CA19.9 (150 U/mouse); (iv) mouse IgG3 (120 �g/mouse); (v) mouse IgG3 � CA19.9; and (vi)phosphate-buffered saline. The immunization interval was 1 week.

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Figure 5. Complement-dependent cytotoxicity induced bymonoclonal antibody (mAb) LC44. Approximately 1 � 104

chromium-labeled SW1116 cells were incubated with mAbLC44 or IgG3 (5 �g/mL) at 37°C for 30 minutes. The cellswere then incubated with T503 medium or T503 mediumcontaining 10 �g/mL of human antimouse antibody at 37°Cfor 30 minutes and then with fresh human serum (40%) for4 hours. The released 51Cr was determined by a �-counting.

ing CA19.9 by forming an immunocomplex andwill most likely remove the immunosuppres-sion induced by the mucin. Apart from this, theformation of an immunocomplex might be ableto change the conformation of antigen, whichrenders essential capture and process by den-dritic cells (DCs) and induces T-cell responsesagainst the tumor antigen. This step is very cru-cial in cellular immunity against cancer, as onepossible mechanism for lack of cytotoxic re-sponse is the inability of DCs to process im-munogenic tumor antigens. Therefore, the ther-apy with mAb LC44 could potentially break theimmunosuppressive process by two mainmechanisms: (i) by removing CA19.9 from thecirculation and consequently reducing the over-all immunosuppression and (ii) if nakedCA19.9 (naturally circulating antigen) is notprocessed by the APC, the presence of a murineantibody (mAb LC44) bound to the antigenwould change not only its distribution, butlikely also its processing and presentation todifferent cellular compartments. Therefore,once the naked CA19.9 is covered by the in-jected antibody, the complex may be processedby APCs and CA19.9-related sequences can bepresented to T-cells. Our cytotoxic experimentstrongly suggests that when complexed with thespecific antibody (mAb LC44), CA19.9 can es-cape retention in the early endosomes in APCand be transported to the late endosomes to beprocessed and presented to T-cells. Therefore,mAb LC44 can be used either (i) as an immunemodifier to target circulating tumor-associatedCA19.9, or (ii) in the form of immune com-plexes with CA19.9 as vaccines.

Although evidence for a circulating Ag-Abcomplex has been discovered in patients withcolorectal cancer, significant increments inmeasurable plasma antigens have been ob-served in most cancer patients.14,22 Thus, ex-ternal Abs become necessary to eliminate theimmunosuppression of antigens. Systemic ad-ministration of external monoclonal antibodieshas already shown clinical promise,23–25 and in-creasing numbers of mAbs are now becomingcomponents of standard treatment regimens.However, their mechanisms of action still re-main to be fully understood. It is well acceptedthat once antibodies bind to antigens, they ini-tiate CDC, bridge natural killer cells or macro-phages to the tumor for ADCC, interfere withtumor-cell growth by blocking survival or in-ducing apoptotic signals, or increase immuno-

genecity by facilitating the uptake and pres-entation of tumor antigens by APCs. In thisstudy, we also evaluated the ability of mAbLC44 to mediate CDC and ADCC. Althoughwe did not find that mAb LC44 could play arole in ADCC (data not shown), our resultsclearly showed that mAb LC44 was extremelyeffective in mediating CDC. Simultaneously,we also observed a direct killing of mAb LC44to CA19.9-positive tumor cells. Similar phe-nomena appeared in the animal study, as mAbLC44 alone and mAb LC44-CA19.9 complexsignificantly extended the survival time ofCA19.9-expressing tumor-bearing mice. How-ever, further mechanical studies need to be per-formed to elucidate a conclusion.

One obstacle in the development of agents forhuman immunotherapy is that there is no avail-able system for in vivo evaluation.26 In this study,we rebuilt an incomplete human immune systemin immunodeficient mice. This complexity intro-duced many difficulties to the analysis of treat-ment data. For example, mice that received mAbor immunocomplex injections collapsed sharply14 days after treatment. This might be due to thecomplete clearance of the treatment agents. How-ever, we cannot exclude another possibility, thatthe human immune system would not functionany longer after this time point. No matter howimperfect this system was, we could still distin-guish the antitumor effect between mAb LC44and non-mAb LC44 treatment groups. The sur-vival time of SCID mice was significantly pro-longed when they received mAb LC44 as well asLC44 � CA19.9, which demonstrates that the in-jection of either an Ab or antigen-Ab complexachieved a comparable effect. Mice administeredwith CA19.9 had a short survival time, which wasconsistent with our in vitro study. This might in-struct us to make principles for selecting patientsfor a future clinical trial: Patients with higher cir-culating levels of CA19.9 may have a better re-sponse to mAb LC44.

CONCLUSIONS

Taken together, we detected the immunosup-pressive effect of CA19.9 and described an an-titumor effect of mAb LC44. Although this Abstill awaits to be humanized and tested in hu-mans, we hope it can become an important com-ponent of the therapeutic arsenal for gastroin-testinal cancer.

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ACKNOWLEDGMENTS

This project was supported by the Chinese Hi-Tech R&D Program of China (No:2002AA412131).

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