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[Cancer Research 63, 987-993, March 1, 2003]
© 2003 American Association for Cancer Research

Experimental Therapeutics

Immunity to the {alpha}(1,3)Galactosyl Epitope Provides Protection in Mice Challenged with Colon Cancer Cells Expressing {alpha}(1,3)Galactosyl-transferase

A Novel Suicide Gene for Cancer Gene Therapy1

Robert C. Unfer, Daniel Hellrung and Charles J. Link, Jr.2

Stoddard Cancer Research Institute, Iowa Methodist Medical Center, Des Moines, Iowa 50309, and Department of Immunobiology, Iowa State University, Ames, Iowa 50011


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human immunity to {alpha}(1,3)Galactosyl epitopes ({alpha}Gal) may provide the means for a successful cancer gene therapy that uses the immune system to identify and to destroy tumor cells expressing the suicide gene {alpha}(1,3)Galactosyltransferase ({alpha}GT). Innate antibody specific for cell surface {alpha}Gal constitutes a high percentage of circulating IgG and IgM immunoglobulins in humans and is the basis for complement-mediated hyperacute xenograft rejection and antibody-dependent cell-mediated cytotoxicity. In humans, the gene for {alpha}GT is mutated, and cells do not express the {alpha}Gal moiety. We hypothesized that human tumor cells induced to express the {alpha}Gal epitope would be killed by the hosts’ innate immunity. Previous in vitro work by our group has demonstrated complement-mediated lysis of {alpha}Gal-transduced human tumor cells in culture by human serum. To induce antibodies to {alpha}Gal in this in vivo study, {alpha}GT knockout mice were used to determine whether immunization with {alpha}Gal could provide protection from challenge with {alpha}Gal-expressing murine MC38 colon cancer cells. Knockout mice were immunized either a single time, or twice, with rabbit RBC. Antibody titers to {alpha}Gal measured by indirect ELISA were significantly higher in mice immunized twice and approached the titers observed in human serum. Anti-{alpha}Gal antibodies were predominantly of the IgG1 and IgG3 subtype. Immunized knockout mice were challenged i.p. with varying doses of {alpha}Gal+ MC38 colon carcinoma cells. Nonimmunized control groups consisting of {alpha}GT knockout mice, and wild-type C57BL/6 mice were challenged as well with MC38 cells. Immunized mice survived and exhibited slower tumor development in comparison to nonimmunized knockout and control mice. This study demonstrates, in vivo, the protective benefit of an immune response to the {alpha}Gal epitope. Our results provide a basis to pursue additional development of this cancer gene therapy strategy.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cancer gene therapy offers a potential replacement or augmentation of traditional cancer treatments, which use invasive or toxic protocols. Suicide genes that encode an enzyme that activates a prodrug into a toxic molecule, or genes that induce apoptosis, have been or are currently being tested in clinical trials for their efficacy in cancer therapy (1, 2, 3) . Gene therapy vaccine technology is under development for several malignancies. Melanoma has been the favored target because it is a very immunogenic tumor. However, the more common and important forms of cancer have minimal or no immunogenicity. We are developing a novel approach to cancer gene therapy that uses a patient’s innate immunity against xenoantigens to identify and destroy tumor cells. The key aspect of this work is to determine whether colon cancer cells that express a xenoantigen can be rejected by an immune response to the antigen. Colon cancer is the third most common form of cancer and third leading cause of death (4) and provides a good target for a successful cancer gene therapy.

Humans possess specific humoral immunity to {alpha}Gal,3 a major xenotransplant antigen. Although human cells do not carry a functional enzyme for the expression of {alpha}Gal epitopes because of a 2-base frameshift gene mutation (5) , there is evidence that suggests that high titer natural antibody to {alpha}Gal is produced in humans because of continuous antigenic stimulation by gastrointestinal bacteria (5, 6, 7) . Clonal B-cell analyses estimated that ~1% of circulating B cells produce anti-{alpha}Gal antibody (8) . The {alpha}GT catalyzes the transfer of galactose from UDP galactose to the N-acetyl-lactosamine acceptors on carbohydrate side chains of glycoproteins and glycolipids to create the {alpha}Gal moiety. The anti-{alpha}Gal immune response is responsible for initiating hyperacute rejection of vascularized xenotransplants, a severe immunological reaction observed in primates. When {alpha}Gal and specific antibody form immune complexes, complement is activated via the classical pathway (9, 10, 11, 12, 13) .

Our interest in {alpha}Gal-mediated destruction of tumor cells was inspired by studies describing lysis of murine retroviral VPCs after exposure to human peritoneal fluid. VPCs have been used for in vivo gene delivery in several cancer gene therapy studies (14 , 15) . Our laboratory and others have demonstrated that antibody and complement in human serum binds {alpha}Gal within 30 min of exposure and induces complement-mediated lysis of VPCs and the viral vectors they produce (16, 17, 18, 19, 20) . Additionally, Collins et al. (21) showed that human fibroblast cells expressing porcine {alpha}GT were destroyed by antibody and complement. To test whether this gene could be used to induce destruction of tumor cells, a truncated version of the murine {alpha}GT was cloned into a retroviral vector backbone and used to transduce human A375 melanoma cells (22) . During in vitro experiments, >90% of transduced A375 cells expressing {alpha}Gal were killed after exposure to human serum. {alpha}Gal-expressing A375 cells were treated for 30 min with human serum and then injected in vivo into athymic nude mice. All experimental mice remained tumor free, whereas control groups developed tumors (22) . Lysis of {alpha}Gal-expressing murine cells by human serum can be blocked by the addition of complement inhibitors (heparin, enoxaparin) or soluble complement receptor 1 (20) . These data demonstrate the key role that complement has in destruction of targets expressing the {alpha}Gal xenoantigen.

Transgenic knockout mice that lack the {alpha}GT gene ({alpha}GT KO) have been produced (23 , 24) and provide an ideal small animal model to study the in vivo immune response against {alpha}Gal epitopes. While not expressing detectable cell surface {alpha}Gal epitopes, these mice can produce low detectable titers of natural anti-{alpha}Gal, possibly from bacterial stimulation (25 , 26) . Immunization with RRBCs results in the production of anti-{alpha}Gal antibody with titers and specificity similar to those observed in humans (27) . In this report we present in vivo data that shows clear protective benefits of an anti-{alpha}Gal immune response, when RRBC-immunized {alpha}GT KO mice are challenged with {alpha}Gal+ tumor cells. These findings have implications for generation of a system to deliver the {alpha}Gal suicide gene to human tumor cells, and making them susceptible to destruction by natural human immunity to {alpha}Gal.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Media.
MC38 colon carcinoma cells are syngeneic for C57BL/6 mice and express the {alpha}Gal antigen on their cellular surface. B16.BL6-2 melanoma cells, a metastatic nonimmunogenic derivative of the B16.F10 cell line, are also syngeneic for C57BL/6 mice and are {alpha}Gal-negative. All cells were maintained at 37°C in a 5% CO2 incubator. The growth medium consisted of DMEM (Invitrogen-Life Technologies, Inc., Carlsbad, CA) supplemented with 10% FBS (D-10; Invitrogen-Life Technologies, Inc.).

Animal Model.
Knockout mice for {alpha}GT ({alpha}GT KO) were received for establishing a breeding colony from Dr. John B. Lowe of the University of Michigan (23) . C57BL/6 mice were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN), and were used as control mice during tumor implantation studies. All animals were cared for under Institutional Animal Care and Use Committee-approved protocol and housed in a contained facility.

Lectin Staining for {alpha}Gal Epitopes.
MC38 and B16.BL6-2 cells were seeded into 30-mm dishes in D-10, incubated at 37°C in 5% CO2, and grown to confluent monolayers. Cell monolayers were washed twice with HBSS and incubated for 15 min at room temperature with a 1:50 dilution in Opti-MEM (Invitrogen-Life Technologies, Inc.) of FITC-labeled Griffonia simplicifolia IB4 (Vector Laboratories, Inc., Burlingame, CA). This lectin has previously been shown to bind specifically to {alpha}Gal epitopes (28 , 29) . The lectin solution was removed, monolayers were washed twice with HBSS, and fresh Opti-MEM was added to cells. Monolayers were observed for lectin binding using a Nikon Diaphot 300 fluorescent microscope (Nikon, Inc., Melville, NY) and photographed using Fuji 1600 Provia color film (Fuji Photo Film Co., Tokyo, Japan).

Complement-mediated Cell Death.
MC38 cells in culture were trypsinized for 2–3 min at 37°C. The trypsin was inactivated with complete culture media (D-10), and cells were collected by centrifugation at 3000 rpm for 5 min at 4°C. Cells were suspended in 200 µl of one of three possible treatment solutions: 50% DMEM and 15% FBS (D-15) in Opti-MEM; 50% fresh human serum in Opti-MEM; or 50% heat-inactivated human serum in Opti-MEM. In addition, cells were incubated at 37°C for 1 h. Treated cells were collected, resuspended in 100 µl of FITC-labeled IB4 lectin (diluted 1:100 in Opti-MEM), and incubated at room temperature for 10 min. One hundred µl of a PI solution (25 µg/ml) diluted in HBSS were added to the cells and incubated for 5 min at room temperature. Cells were collected, resuspended in Opti-MEM, and analyzed by flow cytometry (Coulter Epics Altra Flow Cytometer, Miami, FL).

{alpha}Gal Antigen Immunization.
Eight to 12-week-old {alpha}GT KO mice were used in this study and cared for under an approved animal protocol using American Association of Laboratory Animal Care guidelines. Mice were immunized i.p., with 107 {alpha}Gal+ female NZW RRBCs (Cocalico Biologicals, Inc., Reamstown, PA) suspended in 100 µl of HBSS. Immunizations were given either a single time 14 days before MC38 and B16.BL6-2 tumor cell challenge or twice at 28 days and 14 days before MC38 tumor cell challenge. Control {alpha}GT KO and control C57BL/6 mice were mock immunized with 100 µl of HBSS i.p.

Antibody Titration and Subtyping.
Antibody specific for the {alpha}Gal epitope was detected, and end point was titrated using an indirect ELISA. {alpha}Gal antigen ({alpha}Gal-BSA; V-Labs, Inc., Covington, LA) was diluted to 5 µg/ml in carbonate buffer (pH 9.5) and coated onto polyvinyl chloride (PVC) ELISA plates (Falcon 3912; Becton Dickinson Labware, Franklin Lakes, NJ) overnight at 37°C in a humidified chamber. Nonspecific binding sites in assay wells were blocked for 2 h with a solution of 1% BSA (Fraction V; Sigma) in carbonate buffer. Two-fold serial dilutions of primary sera were made in wash buffer [1x PBS (pH 7.4), 0.05% Tween 20), added to antigen-coated wells, and incubated for 1 h at 37°C. Wells were washed five times, and a secondary antibody, horseradish peroxidase-labeled goat antimouse IgG H+L diluted 1:5000 in wash buffer (Pierce Chemical Co., Rockford, IL), was added to assay wells and incubated 1 h at room temperature. Wells were washed five times, and 100 µl of 3,3',5',5-tetramethylbenzidine liquid substrate (Sigma) was added. After a 15-min incubation at room temperature, the substrate reaction was stopped with 0.5 N H2SO4, and the absorbance at 450 nm for each well was determined using a Molecular Dynamics SpectraMax 250 Plate reader (Sunnyvale, CA).

The murine immune response to {alpha}Gal was measured over time. Serum from immunized mice was diluted 1:100 in wash buffer before detection by ELISA as before. Anti-{alpha}Gal antibody class and subtype was determined from serum collected 14 days after the last RRBC immunization, using a Zymed MonoAb ID ELISA kit (Zymed Laboratories, Inc., San Francisco, CA). {alpha}Gal-BSA antigen was coated onto PVC ELISA plate wells as before, and nonspecific binding sites were blocked using 1% BSA. Diluted primary sera were added and incubated with antigen. Secondary rabbit antimouse isotype horseradish peroxidase-labeled antibodies and 3,3',5',5-tetramethylbenzidine substrate were used for detection.

Tumor Cell Challenge.
In the first experiment, 15 {alpha}GT KO mice were immunized i.p. a single time with 107 RRBC in 100 µl of HBSS 14 days before tumor cell challenge. As experimental controls, 15 {alpha}GT KO mice and 15 syngeneic C57BL/6 mice were mock immunized with HBSS. In a blinded experiment, all mice were divided into three sets, with each set comprised of 5 immunized {alpha}GT KO mice, and 5 mice each of the two control groups. Each set of mice was challenged i.p. with a different dilution of MC38 colon carcinoma cells suspended in Plasma-Lite (Baxter Healthcare Corp., Deerfield, IL). Mice in set A were challenged with 2.5 x 104 MC38 cells. Mice in sets B and C were challenged with 5.0 x 104 and 1.0 x 105 MC38 cells, respectively. Separately, 8 {alpha}GT KO mice were immunized a single time as before with 107 RRBC in HBSS 14 days before i.p. challenge with 1.0 x 105 B16.BL6-2 melanoma cells suspended in Plasma-Lite. A second experiment was designed in which 15 {alpha}GT KO mice were immunized twice with 107 RRBC 28 and 14 days before MC38 tumor cell challenge. Fifteen {alpha}GT KO mice and 15 syngeneic C57BL/6 mice were mock immunized with HBSS and served as experimental controls. All RRBC-immunized and mock-immunized control mice were challenged i.p. with 2.5 x 104 MC38 colon carcinoma cells. In all experiments, mice were observed daily for animal morbidity and palpated for tumor growth.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
FITC-Lectin Staining.
MC38 colon carcinoma and B16.BL6-2 melanoma cells were incubated with a solution of FITC-labeled IB4 lectin to identify the differences between the two cell lines in expression of the {alpha}Gal epitope. FITC staining is prominent along the outer surface membrane of cultured MC38 cells (Fig. 1)Citation because this cell line has a functional {alpha}GT gene and expresses the surface {alpha}Gal moiety that is detected by IB4 lectin binding. B16.BL6-2 melanoma cells do not express the {alpha}Gal moiety because they lack a functional {alpha}GT gene and are not stained with IB4 lectin-FITC.



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  		Fig. 1. Visualization of {alpha}Gal expression in MC38 colon carcinoma cells and lack of expression in {alpha}Gal-negative B16.BL6-2 melanoma cells using FITC-IB4 lectin staining. Cells were incubated with a 1:50 dilution of FITC-IB4 lectin in Opti-MEM at room temperature for 15 min. A and B show MC38 cells, and C and D show B16.BL6-2 cells. FITC-lectin staining of the {alpha}Gal moiety in the outer cell surface membrane demonstrates the activity of the {alpha}GT gene in MC38 cells and lack of gene expression in B16.BL6-2 cells. A and C were photographed using a Nikon DM505 FITC cube, and B and D were photographed under brightfield light using the same FITC cube.

 
Complement-mediated Cell Death.
{alpha}Gal-positive MC38 cells were incubated with culture medium containing no human serum, 50% normal human serum, or 50% heat-inactivated human serum. Flow cytometry of FITC-lectin-stained cells provided total cell counts. In addition, PI uptake by dead cells as a percentage of total cell numbers was used to measure cell death after incubation with the test media. Flow cytometry of cells treated with the three test media demonstrated killing of 98% of {alpha}Gal-positive MC38 cells by media containing human serum with active complement (Fig. 2)Citation . Cells that were incubated with media containing heat-inactivated human serum exhibited a 30% cell death by PI uptake, whereas background uptake by untreated cells was 22%.



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  		Fig. 2. {alpha}Gal-positive MC38 colon carcinoma cells are killed when exposed to serum containing active human complement. MC38 cells were stained with FITC-IB4 lectin and suspended in test media containing no human serum, 50% human serum, and 50% heat-inactivated human serum. After 1-h incubation at 37°C, cells were incubated with a suspension of PI for 5 min at room temperature. Flow cytometry demonstrated that only serum containing active complement was able to kill {alpha}Gal expressing MC38 cells.

 
Antibody Titers and Subtyping.
Antibody titers to the {alpha}Gal epitope from immunized {alpha}GT KO mice and mock-immunized control {alpha}GT KO and C57BL/6 mice were determined by indirect ELISA. Fig. 3Citation shows the results of antibody titration in serum from mice that were immunized either a single time 14 days before serum collection (Fig. 3A)Citation , or mice that were immunized twice, 28 and 14 days, before serum collection (Fig. 3B)Citation . Serum from mock-immunized {alpha}GT KO and C57BL/6 mice exhibited an average A450 absorbance of <0.05 at a 1:50 dilution and was considered negative. This serum was pooled and used to determine background antibody binding. Immune sera with an A450 absorbance of >0.1 above background were considered positive. The average titer of anti-{alpha}Gal antibody in mice immunized one time with RRBC was 1:1600 (Fig. 3A)Citation . Mice that were immunized twice with RRBC had an average titer of 1:8000 (Fig. 3B)Citation . The immune response in {alpha}GT KO mice to {alpha}Gal was determined by collecting serum from mice at various times after RRBC immunization on days 14 and 28. An anamnestic IgG immune response to {alpha}Gal that peaks at 7 days after the second immunization was observed (Fig. 4A)Citation .



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  		Fig. 3. High anti-{alpha}Gal antibody titers are induced in {alpha}GT KO mice after immunization with RRBC. An indirect ELISA using an {alpha}Gal-BSA conjugate as the antigen was used to titrate serum collected from {alpha}GT KO mice that were immunized (A) one time with 107 RRBC or (B) twice with a 14-day interval with 107 RRBC. Serum was collected 2 weeks after completion of immunization. Serum from mock-immunized mice was pooled and used to determine background antibody binding. Immune sera with an A450 absorbance of >0.1 above background were considered positive. The average titer of anti-{alpha}Gal antibody in mice immunized one time with RRBC was 1:1600 (A). Mice that were immunized twice with RRBC had an average titer of 1:8000 (B).

 


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  		Fig. 4. Immune response to {alpha}Gal epitopes peaks at 7 days after a second immunization with {alpha}Gal-positive RRBC. A, mice were immunized with 107 RRBC on days 0 and 14 (indicated by arrows). Blood was collected from the saphenous vein at various times from 4 to 26 days after immunizations were initiated. Serum was analyzed using an indirect ELISA to measure the presence of anti-{alpha}Gal antibodies. An anamnestic response to {alpha}Gal was observed after the second immunization. B, serum from RRBC-immunized mice was used to determine anti-{alpha}Gal antibody isotypes by indirect ELISA. Mice immunized once or twice all produced IgM heavy chain and {kappa} light chain antibodies. The predominant IgG subtypes for both sets of immunized mice were IgG1 and IgG3.

 
Serum samples collected on day 0 from {alpha}GT KO mice immunized once on day -14 (14 mice) or twice on days -28 and -14 (8 mice) were assayed for their antibody isotype. An A450 absorbency > 0.2 for an individual antibody isotype was considered positive. Serum was negative for an antibody isotype and subclass if the A450 absorbance was <0.2. RRBC immunization stimulated the production of IgM, IgG1, and IgG3 antibodies (Fig. 4B)Citation . The predominant subclass was IgG3, which developed in 50% (11 of 22 immunized mice). The IgG1 subclass was observed with higher frequency (36%) then either IgG2a or IgG2b (5 and 13%). All mice immunized regardless of schedule demonstrated high levels of IgM antibody, and no immunized mice demonstrated any IgA antibody isotype. Interestingly, all immunized mice developed antibody with {kappa} light chain, and no {lambda} light chain was found in these mice.

Tumor Cell Challenge.
Fourteen days after RRBC immunization of {alpha}GT KO mice and mock immunization of {alpha}GT KO and C57BL/6 controls, the mice were divided into three groups and implanted i.p. with different dilutions of MC38 colon carcinoma cells in a blinded experiment. Tumor cell dilutions were made, coded, and randomized before injection into mice. All mice were observed daily for tumor development and were euthanized when tumors reached ~1200 mm3, exhibited ascites fluid production, or when the mice were moribund. Survival curves of RRBC-immunized and mock-immunized control mice challenged with the three doses of MC38 tumor cells (2.5 x 104, 5.0 x 104, or 1.0 x 105) are presented (Fig. 5)Citation . Immunized {alpha}GT KO mice were observed to develop tumors more slowly regardless of the amount of MC38 cells used for challenge. Immunized and mock-immunized mice challenged with the three dilutions of MC38 cells were pooled and analyzed as a group. The percentage of mock-immunized control {alpha}GT KO mice (47%) that survived challenge with the three dilutions of MC38 cells was lower than the percentage of RRBC-immunized {alpha}GT KO mice (87%) that were challenged (P = 0.031). All three dilutions of MC38 cells were able to rapidly establish tumors in mock-immunized C57BL/6 control mice, and all C57BL/6 control mice were euthanized by 18 days after tumor cell. In a separate experiment, the survival curve for RRBC-immunized {alpha}GT KO mice challenged with 1 x 105 B16.BL6-2 cells demonstrates that immunity to {alpha}Gal does not protect against challenge with {alpha}Gal- melanoma cancer cells (Fig. 6)Citation . In this experiment, all 8 immunized {alpha}GT KO mice developed tumors rapidly and were euthanized by day 25.



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  		Fig. 5. Mice immunized with RRBC showed superior protection to tumor challenge with {alpha}Gal-positive MC38 colon carcinoma cells compared with mock-immunized mice. Survival curves for immunized {alpha}GT KO mice and mock-immunized {alpha}GT KO mice and C57BL/6 mice after MC38 tumor cell challenge demonstrate that an immune response to {alpha}Gal epitopes can increase the survival of immunized mice in comparison to mice that are not immunized with {alpha}Gal. Group 1 consisted of {alpha}GT KO mice that were immunized one time with 107 RRBC before challenge 14 days later with MC38 cells. Group 2 consisted of {alpha}GT KO mice that were mock immunized and challenged 14 days later. Group 3 consisted of C57BL/6 mice that were mock immunized and challenged 14 days later. Three dilutions of MC38 cells were used to challenge mice (A) 2.5 x 104 MC38 cells, (B) 5.0 x 104 MC38 cells, or (C) 1.0 x 105 MC38 cells. Mock-immunized C57BL/6 mice all died or were euthanized within 18 days. Mock-immunized {alpha}GT KO mice showed 40–60% survival. Immunized {alpha}GT KO mice showed 80–100% survival (P = 0.031).

 


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  		Fig. 6. Mice immunized with RRBC were not protected after tumor challenge with {alpha}Gal-negative B16.BL6-2 melanoma cells. Survival curve for immunized {alpha}GT KO mice after challenge with B16.BL6-2 cells shows that an immune response to {alpha}Gal epitopes does not prevent tumor growth and death. Mice were immunized one time with 107 RRBC and challenged 14 days later with 1.0 x 105 B16.BL6-2 melanoma cells. None of the mice survived tumor challenge despite immunization with {alpha}Gal antigen because the B16.BL6-2 tumor cell line is a non-{alpha}Gal-expressing cell.

 
The challenge experiment was repeated using a single dilution (2.5 x 104) of MC38 tumor cells to challenge {alpha}GT KO mice immunized twice with RRBC to increase their antibody titers and mock-immunized control mice. After tumor cell challenge, all mice were closely monitored and palpated for tumor growth (Fig. 7)Citation . None of the mock-immunized C57BL/6 control mice (0%) survived beyond 17 days after tumor cell challenge. Only 9 of 15 (60%) mock-immunized {alpha}GT KO mice survived beyond 32 days after challenge. In contrast, all of the immunized {alpha}GT KO mice (100%) survived MC38 challenge (P = 0.0069), and palpated tumors were smaller and developed much more slowly when compared with mock-immunized {alpha}GT KO mice.



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  		Fig. 7. Mice immunized twice with RRBC showed superior protection to tumor challenge with {alpha}Gal-positive MC38 colon carcinoma cells compared with mock-immunized mice. Survival curves for immunized {alpha}GT KO mice (group 1) and mock-immunized {alpha}GT KO mice (group 2) and C57BL/6 mice (group 3) after MC38 tumor cell challenge. Mice were immunized or mock immunized twice with 107 RRBC, 28 and 14 days before tumor cell challenge. All mice were challenged with 2.5 x 104 MC38 cells. Group 1 {alpha}GT KO mice were immunized with RRBC and demonstrated 100% survival to tumor cell challenge (P < 0.0069) in comparison to group 2 mock-immunized {alpha}GT KO mice (60% survival) and group 3 mock-immunized C57BL/6 mice (0% survival).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The role of {alpha}Gal epitopes in xenograft rejection has been well studied (9, 10, 11, 12, 13 , 30) . Rapid immune recognition of {alpha}Gal epitopes on the surface of foreign cells results in antibody-mediated cell cytotoxicity and complement-mediated lysis of targeted cells. This immunity is a barrier to the use of nonhuman organs and tissue for transplant purposes. Recent advances in transplant science have resulted in the development of {alpha}GT KO pigs that may overcome immune obstacles to successful xenotransplantation in humans (31) . Cancer gene therapy protocols that rely upon murine VPC for delivery of genes can also be limited by the potent human immune response to {alpha}Gal epitopes. Both VPC and the vectors they produce are destroyed by anti-{alpha}Gal immune mechanisms (16, 17, 18, 19, 20) . Clonal B-cell analysis estimated that ~1% of circulating B cells produce anti-{alpha}Gal antibody (8) , and an estimated 1–2.4% of circulating IgG and 3.9–8% of IgM are specific anti-{alpha}Gal antibodies (32 , 33) . We hypothesize that innate anti-{alpha}Gal immunity that is disadvantageous for xenograft transplantation and VPC therapies could be used as an advantageous method to induce the destruction of cancer cells. Human cancer cells expressing {alpha}Gal epitopes on their cell surface would appear as xenoantigens and induce a strong immune response that destroys them. A murine colon cancer cell line was chosen for this study. Colon cancer is the third most common form of cancer and third leading cause of death (4) .

The {alpha}GT KO mouse is an ideal small animal model to study our hypothesis. The loss of the {alpha}GT gene by these mice mimics the evolutionary loss of this gene by ancestral Old World primates and humans (5) . {alpha}GT KO mice produce little or no {alpha}Gal-specific antibody (25 , 26) but are able to develop an immune response to {alpha}Gal when immunized with RRBCs. These mice can produce {alpha}Gal-specific antibody with high titers and specificity in some animals similar to those observed in humans (27) . The RRBC immunization protocol we used with the {alpha}GT KO mice resulted in high titers of anti-{alpha}Gal antisera that could be detected by indirect ELISA at a 1:16,000 dilution. Previously, LaTemple et al. (34) demonstrated a partially protective immune response when {alpha}GT KO mice are vaccinated with {alpha}Gal-expressing B16 cells (after stable transfection of {alpha}Gal-negative B16 cells with {alpha}GT cDNA) and challenged with parental B16 cells.

MC38 murine colon carcinoma cells have a functional {alpha}GT enzyme and express {alpha}Gal on their cell surface glycoproteins. Fig. 1Citation shows the difference in {alpha}Gal expression between MC38 colon carcinoma and B16.BL6-2 melanoma cells used in this study. The {alpha}Gal moiety expressed on the surface of MC38 cells is labeled brightly with IB4 lectin-FITC conjugates, whereas B16.BL6-2 melanoma cells lack {alpha}Gal expression on their surface and do not bind the IB4 lectin. A serum exposure assay showed the complement-mediated destruction of {alpha}Gal-positive MC38 colon carcinoma cells. Cells were incubated with media that contained 50% human serum with active complement or media that contained 50% heat-inactivated human serum (Fig. 2)Citation . After incubation with the test media, PI uptake measured by flow cytometry was used to estimate the percentage of cells killed. A total of 98% of MC38 cells was killed when exposed to untreated human serum. Control cells that were untreated or treated with heat-inactivated serum (devoid of active complement) showed PI uptake of 22 and 30%, respectively). Therefore, the presence of active human complement induced dramatically higher killing as expected (22) . Takeuchi et al. (18) demonstrated similar results using human cells. Transfected cells that express porcine {alpha}GT are lysed by human serum with complement (18) .

The titer of anti-{alpha}Gal antibody in immunized {alpha}GT KO mice was measured by indirect ELISA. Mice immunized twice with RRBC developed an average titer of 1:8000 (Fig. 3B)Citation that is comparable with measured serum titers from patients receiving VPC treatment (data not shown). Mice that were immunized twice with RRBC developed an anamnestic immune response to the {alpha}Gal antigen (Fig. 4A)Citation . The titer of anti-{alpha}Gal peaked 7 days after the second immunization, and IgG1, IgG3, and IgM are the dominant anti-{alpha}Gal heavy chain isotypes (Fig. 4B)Citation .

Tumor challenge studies were designed to determine whether immunity to {alpha}Gal epitopes could provide protection from challenge with {alpha}Gal-expressing MC38 murine colon carcinoma cells. In the first experiment, {alpha}GT KO mice were immunized a single time with 107 RRBC and challenged with different dilutions (2.5 x 104, 5.0 x 104, or 1.0 x 105) of MC38 cells. A total of 13 of 15 {alpha}GT KO mice survived tumor challenge, whereas only 7 of 15 mock-immunized {alpha}GT KO mice survived challenge with the same dilutions of MC38 cells (P = 0.031). Despite our evidence that mice immunized a single time with RRBC do not develop high titers of anti-{alpha}gal antibody, 87% of immunized mice were protected and survived. None of the mock-immunized control C57BL/6 mice survived the tumor challenge, and all were euthanized by day 18. Although anti-{alpha}Gal antibody could not be detected by ELISA in sera from mock-immunized control mice, others have suggested that {alpha}GT KO mice have a low natural titer of anti-{alpha}gal antibody (25 , 26) . This natural antibody in the transgenic knockout mouse may, as hypothesized in humans, arise from stimulation of environmental antigens. The combination of preexisting low antibody titers and stimulation by {alpha}Gal-positive MC38 cells may have allowed 47% of the mock-immunized {alpha}GT KO mice to survive challenge with the MC38 cell dilutions. In a separate experiment, {alpha}GT KO mice were immunized a single time with 107 RRBC and challenged with 1.0 x 105 {alpha}Gal-negative B16.BL6-2 murine melanoma cells. None of these mice survived beyond day 25 of the tumor challenge (Fig. 6)Citation , and results of this experiment provide evidence that anti-{alpha}Gal antibodies do not provide protection against tumors that do not express the {alpha}GT gene. The potential cellular differences (besides the expression of {alpha}Gal epitopes) between MC38 and B16.BL-2 cells have been addressed by additional experiments in which immunized mice were challenged with B16.BL-2 cells transduced with a vector carrying the {alpha}GT gene. Results show a significant difference in tumor development and kinetics of tumor growth between immunized mice challenged with {alpha}Gal-expressing B16.BL-2 cells and mice challenged with {alpha}Gal-negative B16.BL-2 cells.4 These findings are similar to those reported by LaTemple (34) . A second MC38 challenge experiment incorporated two RRBC immunizations of {alpha}GT KO mice to generate higher titers of anti-Gal antibodies. Immunized and mock-immunized control mice were challenged with 2.5 x 104 of MC38 tumor cells. All 15 immunized {alpha}GT KO mice survived MC38 challenge, whereas only 9 of 15 mock-immunized {alpha}GT KO mice survived (P = 0.0069 by ANOVA). Again, all mock-immunized C57BL/6 control mice developed tumors rapidly and were euthanized by day 17. After the challenge, mice were observed and palpated daily. Observations included slower development of tumors and smaller tumors in immunized {alpha}GT KO mice compared with mock-immunized mice (data not shown). The survival of 60% of the control mock-immunized {alpha}GT KO mice may again be attributable to a combination of low-titer natural antibody and immune stimulation by the MC38 cells. These data provide a first step toward the development of {alpha}Gal-based colon cancer vaccines for humans.

Colorectal cancer vaccines are as yet in the experimental stage of development. Potential vaccines based upon 17-1A, 791Tgp, carcinoembryonic antigen (35) , and the SART3 peptide antigens (36) are currently being tested with marginal but encouraging results. Colon cancer cells expressing a functional {alpha}GT gene will be readily identified by the high percentage of innate circulating human antibodies. This, in turn, would lead to hyperacute rejection of such genetically modified cells. Hyperacute rejection of porcine xenotransplants occurs after recognition and binding of multiple glycoprotein epitopes by human serum (13 , 37) . At least five major cell surface glycoprotein groups on porcine cells express {alpha}Gal epitopes and are detected by human IgM and IgG antibodies and also bind IB4 lectin (38) . Similarly, the MC38 colon carcinoma cells used in this study can also be assumed to present multiple epitopes to the {alpha}GT KO murine immune system.

We have developed an in vivo animal model to demonstrate that immunity to {alpha}Gal can protect {alpha}GT KO mice against challenge with colon cancer cells that express {alpha}Gal. These results demonstrate the potential for a cancer gene therapy that uses the innate immunity to {alpha}Gal antibody in humans. Direct gene transfer of {alpha}GT to in vivo tumors will be the key next step in determining whether {alpha}GT gene therapy for colon cancer will be successful. Gene delivery to tumor cells and expression of the {alpha}GT gene will present multiple targets for the immune system. Because both opsonization and complement fixation are dependent upon epitope density, the potential for a protective immune response directed against human tumor cells that express {alpha}Gal is great.


   ACKNOWLEDGMENTS
 
We thank Dawn Bertrand, Stoddard Cancer Research Institute Animal Care Coordinator, for excellent care of animals and Margaret Liotta-Davis and Marjan Mokhtarian for assistance in blood collection and immunoassays.


   FOOTNOTES
 
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.

1 This work was supported by funding from the United States Department of Defense Grant DAMD17-01-1-0292, and Susan G. Komen Grant 99-3215. Back

2 To whom requests for reprints should be addressed, at Stoddard Cancer Research Institute, Iowa Methodist Medical Center, 1415 Woodland Avenue, Suite 218, Des Moines, Iowa 50309. Phone (515) 241-8787; Fax: (515) 241-8788; E-mail: linkcj{at}ihs.org Back

3 The abbreviations used are: {alpha}Gal, {alpha}(1,3)Galactosyl epitope; {alpha}GT, {alpha}(1,3)Galactosyltransferase enzyme; {alpha}GT KO, {alpha}(1,3)Galactosyltransferase knockout; VPC, vector producer cell; RRBC, rabbit RBC; IB4, isolectin B4; PI, propidium iodide. Back

4 G. Rossi, personal communication. Back

Received 7/26/02. Accepted 12/27/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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