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[Cancer Research 65, 10555-10561, November 15, 2005]
© 2005 American Association for Cancer Research

Immunology

Effective Treatment of Preexisting Melanoma with Whole Cell Vaccines Expressing {alpha}(1,3)-Galactosyl Epitopes

Gabriela R. Rossi1,2, Mario R. Mautino1, Robert C. Unfer2, Tatiana M. Seregina1,2, Nicholas Vahanian1 and Charles J. Link1,2

1 NewLink Genetics Corp., ISU Research Park, Ames, Iowa and 2 Iowa Cancer Research Foundation, Urbandale, Iowa

Requests for reprints: Gabriela R. Rossi, NewLink Genetics Corp., ISU Research Park, 2901 South Loop Drive, Suite 3900, Ames, IA 50010-8646. Phone: 515-296-3269; E-mail: grossi{at}linkp.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The hyperacute immune response in humans is a potent mechanism of xenograft rejection mediated by complement-fixing natural antibodies recognizing {alpha}(1,3)-galactosyl epitopes ({alpha}Gal) not present on human cells. We exploited this immune mechanism to create a whole cell cancer vaccine to treat melanoma tumors. B16 melanoma vaccines genetically engineered to express {alpha}Gal epitopes (B16{alpha}Gal) effectively treated preexisting s.c. and pulmonary {alpha}Gal-negative melanoma (B16Null) tumors in the {alpha}(1,3)-galactosyltransferase knockout mouse model. T cells from mice vaccinated with B16{alpha}Gal recognized B16Null melanoma cells measured by detection of intracellular tumor necrosis factor-{alpha}. We showed successful adoptive transfer of immunity to recipient mice bearing lung melanoma metastasis. Mice receiving lymphocytes from donors previously immunized with B16{alpha}Gal had reduced pulmonary metastases. The transfer of lymphocytes from mice vaccinated with control vaccine had no effect in the pulmonary metastasis burden. This study unequivocally establishes for the first time efficacy in the treatment of preexisting melanoma tumors using whole cell vaccines expressing {alpha}Gal epitopes. Vaccination with B16{alpha}gal induced strong long-lasting cell-mediated antitumor immunity extended to B16Null. These data formed the basis for the testing of this therapeutic strategy in human clinical trials currently under way.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Despite multiple preventive and therapeutic approaches, cancer is one of the major causes of death worldwide. The American Cancer Society estimated >1.3 million new cases of cancer for 2005. Among other therapies, manipulation of the immune system in different types of immunotherapies has shown encouraging results in human clinical trials (1, 2). However, new immunotherapies are greatly needed because currently available treatments are still partially effective in cancer eradication (3). Different modalities of cancer vaccines have shown some degree of clinical efficacy. Whole tumor cell vaccines, administered in presence of adjuvant and genetically engineered tumor cells that express cytokines (i.e., granulocyte macrophage colony-stimulating factor and interleukin 2), are being studied extensively (1, 3). These vaccines have the advantage of expressing relevant tumor-associated antigens shared by the patient's cancer cells. However, one of the disadvantages of these vaccines is the weak antigen presentation and poor ability to stimulate a potent immune response (3). In the present study, we have used a different approach to treat preestablished tumors based on the mechanisms responsible for hyperacute rejection of xenotransplants.

The major barrier for xenotransplantation to humans is the {alpha}(1,3)-galactosyl epitope ({alpha}Gal). The gene encoding {alpha}(1,3)-galactosyltransferase ({alpha}GT), which catalyzes the synthesis of {alpha}Gal epitopes on glycoproteins and glycolipids, is inactive in humans and Old World monkeys but is functional in other mammalian cells (4, 5). The human immune system is continuously stimulated by intestinal and pulmonary bacterial flora to produce natural antibodies that recognize {alpha}Gal epitopes. These anti-{alpha}Gal antibodies initiate a "hyperacute rejection" on tissues and cells expressing {alpha}Gal. The hyperacute rejection of a xenotransplant is characterized by a rapid and acute organ destruction occurring within minutes to hours after transplantation. This reaction is mediated by the binding of anti-{alpha}Gal antibodies from the recipient to {alpha}Gal epitopes expressed on the xenograft and complement activation through the classic pathway (5). In addition, noncomplement fixing natural anti-{alpha}Gal antibody induces antibody-dependent cell-mediated cytotoxicity that initiates tissue damage in xenotransplants mediated by natural killer cells (6–9).

We and others have suggested that the hyperacute rejection of whole cell cancer vaccines expressing {alpha}Gal epitopes could be exploited as new therapeutic approach to treat human malignancies (10–14).

The hypothesis that humoral immunity to {alpha}Gal epitopes may induce anticancer immunity was tested using the {alpha}(1,3)-galactosyltranferase knockout ({alpha}GT KO) mouse model (15). These mice do not express {alpha}Gal epitopes in their cell surfaces and can be induced to develop high titers of anti-{alpha}Gal antibodies (16, 17). We and others have shown that mice with anti-{alpha}Gal antibodies are protected when challenged with {alpha}Gal-expressing cancer cells, whereas no protection was observed in mice without anti-{alpha}Gal antibodies (13, 18). Moreover, the rejection of melanoma cells expressing {alpha}Gal epitopes conferred protection against melanoma cells lacking the expression of {alpha}Gal epitopes. Mice that rejected the first challenge with live B16{alpha}Gal cells were protected from a second rechallenge with B16Null. Moreover, strong CTLs were induced in melanoma protected mice recognizing B16Null (14). In addition, vaccination with B16 melanoma cells expressing {alpha}Gal epitopes prevented tumor development (19).

These data supports the hypothesis that cancer vaccines designed to express {alpha}Gal epitopes could induce strong antitumor immunity. Undoubtedly, the key challenge for the future clinical use of this anticancer vaccine is effective treatment of preestablished tumors.

In this study, we show for the first time that mice with preexisting s.c. melanoma tumors or pulmonary melanoma metastases treated with melanoma vaccine cells expressing {alpha}Gal epitopes exhibited greater survival and fewer pulmonary metastases. Evidence presented here shows that the rejection of {alpha}Gal expressing melanoma vaccine cells in mice with preexisting humoral immunity against this epitope, induces strong, T cell–mediated antitumor immunity that recognizes native {alpha}Gal-negative melanoma cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines. The B16.BL6 melanoma cell line (referred here as B16Null, H-2 b/b) does not express {alpha}Gal epitopes (20). The {alpha}Gal-negative CA320M (H-2 b/b) is a colon carcinoma chemically induced in {alpha}GT KO mouse generated and generously provided by Daniel Hellrung from the Iowa Cancer Research Foundation. B16.BL6 cells were transduced with pLNCKG, encoding the {alpha}GT gene under control of the cytomegalovirus (CMV) promoter (LTR-NeoR-CMV-{alpha}GT-LTR). The expression of {alpha}Gal epitopes was detected in transduced cells by staining with the FITC-labeled Griffonia simplicifolia isolectin B4 (IB4) as previously described (14, 18). B16.BL6 cells transduced to express {alpha}Gal epitopes are referred here as B16{alpha}Gal cells. B16.BL6 cells transduced with pLNL (LTR-NeoR-LTR) yielded {alpha}Gal-negative control cells referred as B16NeoR cells. The pLNCKG vector was provided by Dr. Won-Bin Young (NewLink Genetics, Ames, IA). The activity of {alpha}GT was determined by fluorescence-activated cell sorting (FACS). No {alpha}Gal epitopes were detected in B16NeoR cells and B16Null cells. About 60% to 80% of B16{alpha}Gal cells express {alpha}Gal epitopes (data not shown). All cells were maintained at 37°C in a 5% CO2 incubator in complete medium consisting of DMEM supplemented with 10% fetal bovine serum (FBS), L-glutamine, sodium pyruvate, penicillin, streptomycin, HEPES, 2-ME, and nonessential amino acids. Cell vaccines were prepared by {gamma}-irradiation (200 Gy) of transduced or nontransduced cells.

Mouse and animal treatments. Female and male {alpha}GT KO mice of H-2 b/b haplotype, 8 to 14 weeks old, were used in this study (15). The original {alpha}GT KO mouse expressed both alleles H-2 b and d haplotypes. These animals were generated by crossing C57BL/6 x DAB/2J x 129sv mice (H-2b x H-2d). In an effort to obtain homozygous colonies for the H-2 loci, by breeding and selection, we generated two {alpha}GT KO mouse colonies homozygous for both H-2 b/b and H-2 d/d haplotypes. Animals used in this study express H-2 Kb and H-2Db haplotypes and do not express H-2Dd or H-2Kd (14).

All mice were immunized i.p. with 1 x 108 rabbit RBC (RRBC) 2 or 3 weeks apart to generate anti-{alpha}Gal antibodies as previously described (18). All mice developed high titers (>1:200) of anti-{alpha}Gal antibodies (IgG and IgM) measured by ELISA as previously shown (18). One week following the last RRBC immunization, mice were injected s.c. with 1 x 105 B16Null and randomized. Starting on days 3 to 5 after tumor injection, mice received two or three doses of 5 x 105 irradiated whole cell vaccines 1 week apart from each other (treatment of preestablished s.c. tumors). Developing tumors were measured twice weekly in a blinded manner.

For the treatment of preestablished pulmonary melanoma metastases, mice were injected i.v. with 1 to 5 x 105 B16Null cells. After 4, 11, or 21 days of tumor establishment, mice received either B16{alpha}Gal or B16NeoR vaccines. Each vaccine consisted of 1 to 5 x 105 irradiated cells, administered s.c. Mice were euthanized after 30 days, and pulmonary tumors were enumerated in a blinded manner. All animal experiments were conducted according to the Institutional Animal Care and Use Committee–approved protocols. Histopathology sections of s.c. tumors and lung tissue were stained with H&E.

In vitro detection of T-cell reactivity and adoptive cell transfer experiments. Mice were preimmunized by i.p. injections of RRBC and subsequently vaccinated with three s.c. injections of 2 x 105 irradiated B16NeoR or B16{alpha}Gal cells 1 week apart. Spleen mononuclear cells (Spm) were harvested as previously described (14) and stored in freezing medium (90% FBS and 10% DMSO) in liquid nitrogen until used. Spm cells were rapidly thawed, washed with complete media, and either used for in vitro recognition studies or as donor lymphocytes during adoptive cell transfer experiments. CD8+ T cells were isolated by magnetic purification from Spm using Dynal mouse CD8-negative isolation kit (Oslo, Norway) following manufacturer's instructions. Efficacy of purification was determined by FACS staining with an anti-CD8 monoclonal antibodies (BD PharMingen, San Diego, CA). After purification, cell preparation consisted of >90% CD8+ T cells and >95% CD3+ T cells (data not shown).

Intracellular tumor necrosis factor-{alpha} (TNF-{alpha}) was detected using a mouse detection kit (BD PharMingen) following manufacturer's instructions. Briefly, 1 x 105 target cells/mL (CA320M or B16Null) were cultured overnight with 100 ng/mL of IFN-{gamma} to up-regulate MHC class I (data not shown). After washing target cells to remove nonadherent cells, effector cells (Spm or purified CD8+ T cells) were added to the culture for activation and cytokine production. After 6 hours of incubation in presence of Golgi Plug (BD PharMingen), effector cells were harvested and stained for intracellular TNF-{alpha} and CD3 expression. Up-regulation of cellular activation markers was measured after 24 hours of culture in the absence of brefeldin A. Cells were harvested and stained with PE-labeled anti-CD25 or anti-CD69 monoclonal antibody (BD PharMingen). Acquisition was done using Coulter flow cytometer Epics Ultra (Miami, FL) using Expo32 Software.

For cell transfer experiments, recipients {alpha}GT KO mice were i.v injected with 1 x 105 B16Null cell and randomized 4 to 5 days before cell transfer. They received 1 x 107 donor Spm administered i.p or 1 x 106 purified CD8+ T cells administered i.v. Pulmonary melanoma burden was determined 25 days after tumor inoculation.

Statistical analysis. GraphPad Prism software was used for statistical analysis. Kaplan-Meier survival analysis and log-rank tests were used for curve comparisons. One-way ANOVA test was used when appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effective treatment of established s.c. melanoma tumors by vaccination with B16{alpha}Gal. We first determined whether B16{alpha}Gal vaccination could retard the growth of preexisting s.c. melanoma tumors. Tumors from mice vaccinated with B16{alpha}Gal had significantly reduced growth rate compared with the growth rate of tumors from control mice (Fig. 1A). More than 2-fold reduction in the average of the tumor size was observed at 30 days after challenge following B16{alpha}Gal treatment. Vaccination with B16NeoR had no effect on tumor growth rate. Histopathologic examination of s.c. tumors showed noticeable differences in the quantities and quality of inflammatory cellular infiltrates. Melanoma tumors from nonvaccinated and mice vaccinated with control vaccines (B16Null and B16NeoR) showed minor cellular infiltrates at the edges of the tumor, with only scant lymphocytes around i.t. vessels (Fig. 1B) and no visible granulocytic cells. Tumors from B16{alpha}Gal-vaccinated mice showed mononuclear cell infiltrates at the edges of the tumor and infiltrates within the tumor, consisting of granulocytes, mast cells, and eosinophils. Numerous perivascular lymphocytes were found within the {alpha}Gal-vaccinated tumor mass (Fig. 1C and D).



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  		Figure 1. Teatment of preestablished s.c. melanoma by vaccination with B16{alpha}Gal. A, mice were challenged s.c with B16.BL6 (B16Null) and randomized. Three days after the challenge, they received no treatment (n = 11, {blacksquare}) or were vaccinated twice 7 days apart, with B16NeoR (n = 24, {blacktriangleup}) or with B16{alpha}Gal (n = 24, {bullet}). Tumor sizes were determined by measuring three perpendicular diameters. Tumor growth over time is plotted for each group. Points, mean; bars, ±SE. Slope comparison using straight line model B16NeoR versus B16{alpha}Gal groups (P = 0.0054) and no treatment versus B16NeoR (P = 0.84). B, tumors from control-vaccinated mice had minimal infiltrates of lymphocytes at the periphery of the tumor masses and few lymphocytes around blood vessels that permeated throughout the mass. Some areas showed active proliferation of melanoma cells (arrowhead). C, tumors from mice receiving B16{alpha}Gal vaccines had increased infiltrates around blood vessels that permeated the mass. These infiltrates were present in the vascular adventitia and extended a short distance between tumor cells. In some areas, tumors from B16{alpha}Gal-vaccinated mice had intense infiltrates of lymphocytes mixed with degenerate and/or necrotic neoplastic cells (C and D, arrows). Abbreviations: MEL, melanoma cells; BV, blood vessels; TIL, tumor-infiltrating lymphocytes.

 
Mice vaccinated with B16{alpha}Gal cells had significantly prolonged survival when compared with mice receiving no vaccination of B16NeoR control vaccine (Fig. 2A). Five of 19 mice treated with B16{alpha}Gal vaccine remained tumor free for 70 days. In contrast, only 1 of 20 control mice survived the s.c. challenge with melanoma. The median survival time of the B16{alpha}Gal vaccine group was increased to 39 days compared with only 27 and 29 days for respective controls.



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  		Figure 2. Survival analysis of mice bearing s.c. melanoma treated with B16{alpha}Gal vaccine. A, mice were challenged s.c with B16Null. On days 3 and 10 after challenge, they received either no treatment (n = 9, {blacksquare}) or were vaccinated with B16NeoR (n = 20, {blacktriangleup}) or with B16{alpha}Gal (n = 19, {bullet}). Log-rank test for mice vaccinated with B16NeoR compared with no treatment mice (P = 0.84). Log-rank test for B16NeoR mice compared with B16{alpha}Gal-treated mice (P = 0.02). B, mice were challenged s.c with B16Null. On days 3, 10, and 17 after challenge, they received either no treatment (n = 12, {blacksquare}) or were vaccinated with B16NeoR (n = 23, {blacktriangleup}) or B16{alpha}Gal (n = 26, {bullet}). Log-rank test for no treatment compared with B16NeoR vaccine (P = 0.43). Log-rank test for all group comparison (P = 0.0045).

 
The efficacy of B16{alpha}Gal vaccines was improved by increasing the number of vaccine doses administered in a period of 3 weeks after tumor establishment. As before, mice receiving B16{alpha}Gal vaccines developed smaller tumors that grew significantly slower, whereas tumor growth rates were very similar in control mice (data not shown).

Treatment with three doses of the B16{alpha}Gal vaccine significantly increased the survival of treated mice, whereas vaccination with B16NeoR had no effect on the survival of mice compared with nonvaccinated mice (Fig. 2B). Importantly, surviving mice that received the B16{alpha}Gal vaccine were tumor free at the time the experiment was terminated. These data showed that {alpha}Gal-expressing vaccines induce efficient regression of preexisting s.c. melanoma tumors.

Effective treatment of pulmonary melanoma metastases by vaccination with B16{alpha}Gal. We next evaluated the efficacy of B16{alpha}Gal vaccine for the treatment of pulmonary metastases as a model of disseminated disease. This experiment is very relevant from the clinical point of view, because cancer patients usually die from disseminated nonresectable disease. Three mice treated with B16NeoR vaccine had lungs with "too-numerous-to-count" (arbitrary value, >200) tumors, one mouse had 30 pulmonary metastases, and two mice were tumor free (Fig. 3A). Moreover, one mouse in this control group had three additional metastatic tumors in the peritoneal cavity showing further dissemination of melanoma cells outside the lung tissue. In striking contrast, all mice treated with B16{alpha}Gal vaccine were tumor free by macroscopic observation (Fig. 3B). Histopathologic examination of lungs from B16{alpha}Gal-vaccinated mice revealed no evidence of micrometastases and absence of residual abnormal inflammatory lesions. This suggests the clearance of lung metastasis early after establishment of the pulmonary tumors.



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  		Figure 3. Reduction of lung melanoma metastases by treatment with B16{alpha}Gal vaccine. Pulmonary metastases were established by i.v. injection of B6Null cells. Mice received either 105 (A and B) or 5 x 105 (C) B16Null cells. Mice were randomized and vaccinated with 5 x 105 irradiated B16NeoR or B16{alpha}Gal vaccines on days 4, 11, and 21 after challenge. Thirty days following B16Null challenge, pulmonary melanoma metastases burden was determined. A, mice receiving the B16NeoR vaccine. B, mice treated with B16{alpha}Gal vaccine. C, lungs weighed in block. No vaccine (n = 10), B16NeoR (n = 11), and B16{alpha}Gal (n = 11) vaccines. Bars, SE. *, P < 0.02 (one-way ANOVA).

 
To increase the stringency of these experiments, the i.v. dose of B16Null challenge was increased 5-fold. Numerous pulmonary melanoma metastases developed in the lungs of control groups receiving no treatment or B16NeoR control vaccine. On the contrary, a significant reduction of pulmonary metastasis was shown in mice treated with B16{alpha}Gal vaccine (Fig. 3C).

These data provide strong evidence that the B16{alpha}Gal vaccine can effectively treat preexisting disseminated metastatic melanoma. Furthermore, the vaccination with B16{alpha}Gal prevented spreading of melanoma metastasis outside of the lung cavity.

Induction of T-cell precursors specifically recognizing B16Null after vaccination with B16{alpha}Gal. A fundamental question concerning this novel anticancer vaccine is whether or not T cell–mediated antitumor immunity to the {alpha}Gal-negative melanoma tumor is generated. We addressed this question by harvesting Spm cells from mice vaccinated with B16{alpha}Gal or vaccinated with B16Null and testing their reactivity against B16Null. Purified CD8+ T cells were used as effector cells and cultured with CA320M or B16Null as target cells. We detected significant amount of double-positive cells (TNF-{alpha}+CD3+ cells) in cultures using purified CD8+ T cells from mice vaccinated with B16{alpha}Gal stimulated with B16Null (Fig. 4A, top right; 5.3% TNF-{alpha}+CD3+ cells). Intracellular TNF-{alpha} was not detected when purified CD8+ T cells from B16{alpha}Gal-vaccinated mice were stimulated with a nonmelanoma {alpha}Gal-negative cancer cell line (CA320M, H-2 b/b; Fig. 4A, top left; 0.3% TNF-{alpha}+CD3+ cells), indicating specific recognition of melanoma targets. Only background intracellular TNF-{alpha} staining was observed when purified CD8+ T cells from control vaccinated mice were cultured with either B16Null (0.3% TNF-{alpha}+CD3+ cells) or CA320M (0.7% TNF-{alpha}+CD3+ cells) as targets (Fig. 4A, bottom right and left, respectively). These results indicate that the observed T-cell reactivity is melanoma specific and only induced when mice are vaccinated with {alpha}Gal-expressing melanoma vaccines. Similarly, Spm cells from mice vaccinated with B16NeoR produced very low intracellular TNF-{alpha} when stimulated with B16Null cells (Fig. 4B; mean fluorescence intensity, MFI = 17). In contrast, Spm from B16{alpha}Gal-vaccinated mice exhibited a 4-fold increase in TNF-{alpha} after stimulation with B16Null cells (Fig. 4B; MFI = 69). Spm from mice vaccinated with B16{alpha}Gal and cultured with B16Null cells had significantly increased up-regulation of cell surface activation markers CD25 and CD69 (Fig. 4C and D). This activation was not observed when these Spm were cultured with control CA320M cells. These results show that the up-regulation of T-cell activation molecules was due to specific recognition and activation of cultured lymphocytes by B16Null cells. Control mice vaccinated with B16NeoR and stimulated with B16Null cells had fewer activated splenocytes (Fig. 4C and D). These results show for the first time the induction of melanoma-specific T cells that can recognize {alpha}Gal-negative tumor cells after vaccination with B16{alpha}Gal.



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  		Figure 4. Vaccination with B16{alpha}Gal induces T-cell precursors that recognize B16Null. Purified CD8+ T cells (A) or total spleen mononuclear cells (B-D) were harvested from mice vaccinated with B16{alpha}Gal ({alpha}Gal T, black columns) or with B16Null (Control T, gray columns) to measure in vitro recognition of B16Null and T-cell activation through detection of intracellular TNF-{alpha} by FACS (A-B) or through up regulation of CD25 (C) and CD69 (D) activation markers. Incubation of effector T cells with the {alpha}Gal-negative CA320M (H-2 b/b) was used as non–melanoma-negative control. Repeated twice with similar results using pooled splenocytes or purified T cells from three mice in each group.

 
Antitumor immunity adoptively transferred to syngeneic recipients bearing melanoma metastasis. To confirm that the presentation of {alpha}Gal epitopes on whole cell cancer vaccines induces T cells that recognize melanoma cells lacking the expression of {alpha}Gal epitopes, we did adoptive T-cell transfer experiments. A significant reduction of pulmonary melanoma metastases was observed in mice receiving total spleen mononuclear (Spm) cells harvested from B16{alpha}Gal-vaccinated mice (Fig. 5A). The transfer of SPm cells from B16NeoR-vaccinated mice had no effect on the pulmonary tumor burden (Fig. 5A). To confirm that CD8+ T cells with antitumor activity are induced by B16{alpha}Gal vaccines, CD8+ cells were purified and transferred into mice bearing 5-day pulmonary metastases (Fig. 5A). Mice receiving no CD8+ cells or receiving purified CD8+ T cells from mice vaccinated with B16Null control vaccine had significant numbers of pulmonary metastases. In striking contrast, 11 of 12 mice receiving CD8+ T cells from mice vaccinated with B16{alpha}Gal were free of pulmonary tumors, and 1 of 12 had only a small number of small tumors (Fig. 5B).



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  		Figure 5. Cell-mediated antitumor immunity was adoptively transferred to syngeneic mice. A, donor mice received RRBC immunizations and three s.c. injections of 5 x 105 B16NeoR, or B16{alpha}Gal vaccine cells 1 week apart. Two weeks following the final vaccination, Spm cells were harvested and transferred to syngeneic recipient mice (1 x 107 cells per mouse, administered i.p.). Four days before cell transfer, recipient mice were injected i.v with B16Null melanoma. Recipient mice received either no Spm cells (white column, n = 17), Spm from mice vaccinated with B16NeoR (gray columns, n = 15 control Spm), or Spm cells from mice vaccinated with B16{alpha}Gal (black column, n = 17 {alpha}Gal Spm). Columns, mean weight of lungs obtained in block; bars, SE. *, P < 0.05 (one-way ANOVA). Similar results were obtained in a second independent experiment (data not shown). B, CD8+ T cells from mice vaccinated with control (B16Null) or B16{alpha}Gal vaccines were purified and transferred to syngeneic recipients bearing 5 days pulmonary melanoma metastasis (1 x 106 purified CD8+ T cells per mouse, administered i.v). Control mice received no CD8+ T cells.

 
The significant reduction of established pulmonary melanoma metastasis by adoptive transfer of CD8+ T cells from B16{alpha}Gal-vaccinated mice provides further evidence for the induction of a strong, specific CD8+ T cell–mediated antitumor immunity by {alpha}Gal+ melanoma vaccines. The results suggest that the presence of {alpha}Gal epitopes on cell surface proteins and lipids of melanoma cells function as a strong immune adjuvant able to induce cell-mediated antitumor immunity against melanoma cells lacking the expression of {alpha}Gal epitopes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present report, we clearly show for the first time efficacy in the treatment of established melanoma tumors using a novel concept of immunotherapy based on hyperacute rejection of whole cancer vaccine cells expressing {alpha}Gal xenoepitopes.

Mice with either preexisting s.c. tumors or pulmonary melanoma metastases were successfully treated with {alpha}Gal-expressing melanoma vaccines. This vaccine preparation differed from control vaccines only by its glycosylation pattern. Without other external cytokines and/or previously described adjuvants, the combination of {alpha}Gal epitopes on vaccine cells and preexisting anti-{alpha}Gal antibodies triggered a cascade of events that ultimately concludes in a potent cell-mediated antitumor immune response. T cells generated by this mean recognized native melanoma cells lacking the expression of {alpha}Gal epitopes, indicating that the specificity of the immune reaction was extended to {alpha}Gal-negative target antigens. This concept of whole cell vaccination differs from other similar studies in several mechanistic steps. First, it takes advantage of preexisting antibodies to the whole cell vaccine, favoring uptake and presentation of tumor-associated antigens. Second, complement activation results in the recruitment and maturation of antigen-presenting cells. Third, antigen uptake relies on Fc receptor–mediated phagocytosis. These elements combined together are unique compared with other whole cell vaccines previously described.

With results presented here and other published information, we propose a model that describes some elements important for this vaccine to generate such strong antitumor immunity (Fig. 6). Allogeneic or autologous tumor cells (from human or {alpha}GT KO mice origin) genetically engineered to express the {alpha}GT enzyme express {alpha}Gal epitopes in multiple proteins and lipids that traffic through the Golgi apparatus. These {alpha}Gal-coated cells constitute a multivalent vaccine containing several {alpha}Gal molecules and multiple tumor-associated antigens, which can be {alpha}-galactosylated or not. Glycosphingolipids and glycoproteins are potential tumor antigens (e.g., melanoma-specific tyrosinase and gp100; ref. 21). Immediately after the injection of {alpha}Gal-positive whole cell vaccines in {alpha}Gal-negative subjects with high titers of anti-{alpha}Gal antibodies, such as humans or {alpha}GT KO mouse, vaccine cells are opsonized by these anti-{alpha}Gal antibodies. Antibody opsonization of {alpha}Gal epitopes induces complement activation through the classic pathway and cell lysis (22). Also opsonization promotes Fc receptor–mediated phagocytosis (23), which is a very efficient mechanism of antigen uptake and processing important for the cross-presentation of antigens to CD8+ T cells (24, 25). Complement-mediated cell lysis generates a "danger signal," which has numerous implications for the kind of immune response that will be generated at the injection site (26, 27). Danger signals (necrosis, tissue injury, and release of heat shock proteins) are recognized as crucial components for antigen-presenting cell activation and differentiation to mature dendritic cells. Cell debris for example, acts as immune adjuvant by releasing among others, heat shock proteins. These molecules activate antigen-presenting cells through TOLL-like receptors. Antigen-presenting cells activated by these receptors are empowered for efficient T-cell activation (28, 29). In addition, complement degradation products have chemoattractant properties that result in recruitment of macrophages, granulocytes, and eosinophils (data not shown). Moreover, natural antibodies and complement are endogenous adjuvants for vaccine-induced CD8+ T-cell responses (30). Two main mechanisms of antigen uptake take place during this phase of hyperacute rejection. The exogenous pathway, which results in presentation of the processed antigen on MHC class II surface molecules and activation of CD4+ T helper cells (31), and the uptake of immunocomplexes involving Fc receptors and anti-{alpha}Gal antibodies. This will favor the cross-presentation pathway resulting in antigen presentation in the context of MHC class I molecules that will preferentially activate CD8+ cytotoxic T cells (24, 25, 32, 33). These cascade of events will ultimate induce activation, proliferation, and expansion of T cells recognizing tumor antigens expressed in tumor cells lacking the {alpha}GT enzyme (Figs. 4 and 5). These cells are able to migrate to the tumor site and infiltrate and eradicate preestablished tumors (Figs. 1–3).



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  		Figure 6. Model proposed for induction of cell-mediated antitumor immunity by hyperacute rejection of {alpha}Gal-expressing vaccines. 1, transduction of {alpha}Gal-negative tumor cells (humans or from {alpha}GT KO mice) to express the {alpha}GT enzyme generates a multivalent {alpha}Gal+ vaccine. 2, opsonization of {alpha}Gal+ whole cell vaccines in {alpha}Gal– subjects with high titers of anti-{alpha}Gal antibodies. 3, antibody opsonization of {alpha}Gal epitopes induces complement activation through the classic pathway and cell lysis. In addition, opsonization promotes Fc receptor–mediated phagocytosis. 4, complement-mediated cell lysis generates a "danger signal" (necrosis, tissue injury, and release of heat shock proteins) that induce activation and differentiation of antigen-presenting cells to mature dendritic cells. Macrophages (Mac), granulocytes (Gr), and eosinophils (Eo) are recruited to the site of inflammation. 5, two mechanisms of antigen uptake result in the presentation of the processed antigen on MHC class II and class I molecules and activation of CD4+ T helper cells and CD8+ cytotoxic T cells, respectively. CD1-restricted T cells may be also activated. 6, T cells recognizing tumor antigens expressed in tumor cells lacking the {alpha}GT enzyme proliferate. 7, these CD8+ T cells migrate within the {alpha}Gal-negative tumor mass and eradicate preestablished tumors. Abbreviations: Gp, glycoproteins; GL, glycolipids; ER, endoplasmic reticulum; DC, dendritic cells; TAA, tumor-associated antigens.

 
Data presented here shows that this approach is effective for the treatment of established melanoma tumors. Based on this and other studies by our laboratory, phase I/II clinical trials using genetically modified {alpha}Gal-positive human allogeneic cancer vaccines for the treatment of human cancer were Food and Drug Administration approved and are currently open.


   Acknowledgments
 
Grant support: Newlink Genetics Corp. Research and Development Agreement (G.R. Rossi).

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.

We thank Dawn Bertrand for her outstanding efforts and invaluable help in the management of the Animal Care Facility at the Iowa Cancer Research Foundation, Dr. Sergei Kisselev for his assistance in the FACS analysis, and Dr. Mark R. Ackermann for his help in the interpretation of histology samples.


   Footnotes
 
Note: G.R. Rossi, M.R. Mautino, N. Vahanian, T.M. Seregina, and C.J. Link have financial Interest in NewLink Genetics Corp.

Received 2/22/05. Revised 7/24/05. Accepted 9/ 9/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Berzofsky JA, Terabe M, Oh S, et al. Progress on new vaccine strategies for the immunotherapy and prevention of cancer. J Clin Invest 2004;113:1515–25.[CrossRef][Medline]
  2. Waldmann TA. Immunotherapy: past, present and future. Nat Med 2003;9:269–77.[CrossRef][Medline]
  3. Finn OJ. Cancer vaccines: between the idea and the reality. Nat Rev Immunol 2003;3:630–41.[CrossRef][Medline]
  4. Larsen RD, Rivera-Marrero CA, Ernst LK, Cummings RD, Lowe JB. Frameshift and nonsense mutations in a human genomic sequence homologous to a murine UDP-gal:ß-D-gal(1,4)-D-GlcNAc {alpha}(1,3)- galactosyltransferase cDNA. J Biol Chem 1990;265:7055–61.[Abstract/Free Full Text]
  5. Joziasse DH, Oriol R. Xenotransplantation: the importance of the Gal{alpha}1,3Gal epitope in hyperacute vascular rejection. Biochim Biophys Acta 1999;1455:403–18.[Medline]
  6. Baumann BC, Forte P, Hawley RJ, Rieben R, Schneider MK, Seebach JD. Lack of galactose-{alpha}-1,3-galactose expression on porcine endothelial cells prevents complement-induced lysis but not direct xenogeneic NK cytotoxicity. J Immunol 2004;172:6460–7.[Abstract/Free Full Text]
  7. Schaapherder AF, Daha MR, te Bulte MT, van der Woude FJ, Gooszen HG. Antibody-dependent cell-mediated cytotoxicity against porcine endothelium induced by a majority of human sera. Transplantation 1994;57:1376–82.[Medline]
  8. Watier H, Guillaumin JM, Vallee I, et al. Human NK cell-mediated direct and IgG-dependent cytotoxicity against xenogeneic porcine endothelial cells. Transpl Immunol 1996;4:293–9.[CrossRef][Medline]
  9. Watier H, Guillaumin JM, Piller F, et al. Removal of terminal {alpha}-galactosyl residues from xenogeneic porcine endothelial cells. Decrease in complement-mediated cytotoxicity but persistence of IgG1-mediated antibody-dependent cell-mediated cytotoxicity. Transplantation 1996;62:105–13.[CrossRef][Medline]
  10. Link CJ, Jr., Seregina T, Atchison R, Hall A, Muldoon R, Levy JP. Eliciting hyperacute xenograft response to treat human cancer: {alpha}(1,3) galactosyltransferase gene therapy. Anticancer Res 1998;18:2301–8.[Medline]
  11. Galili U, LaTemple DC. Natural anti-Gal antibody as a universal augmenter of autologous tumor vaccine immunogenicity. Immunol Today 1997;18:281–5.[Medline]
  12. Galili U. Autologous tumor vaccines processed to express {alpha}-gal epitopes: a practical approach to immunotherapy in cancer. Cancer Immunol Immunother 2004;53:935–45.[Medline]
  13. Posekany KJ, Pittman HK, Swanson MS, Haisch CE, Verbanac KM. Suppression of Lewis lung tumor development in {alpha} 1,3 galactosyltransferase knock-out mice. Anticancer Res 2004;24:605–12.[Abstract/Free Full Text]
  14. Rossi GR, Unfer RC, Seregina T, Link CJ. Complete protection against melanoma in absence of autoimmune depigmentation after rejection of melanoma cells expressing {alpha}(1,3)galactosyl epitopes. Cancer Immunol Immunother 2005;54:999–1009.[CrossRef][Medline]
  15. Thall AD, Maly P, Lowe JB. Oocyte Gal {alpha} 1,3Gal epitopes implicated in sperm adhesion to the zona pellucida glycoprotein ZP3 are not required for fertilization in the mouse. J Biol Chem 1995;270:21437–40.[Abstract/Free Full Text]
  16. Xu H, Sharma A, Chen L, et al. The structure of anti-Gal immunoglobulin genes in naive and stimulated Gal knockout mice. Transplantation 2001;72:1817–25.[CrossRef][Medline]
  17. Posekany KJ, Pittman HK, Bradfield JF, Haisch CE, Verbanac KM. Induction of cytolytic anti-Gal antibodies in {alpha}-1,3-galactosyltransferase gene knockout mice by oral inoculation with Escherichia coli O86:B7 bacteria. Infect Immun 2002;70:6215–22.[Abstract/Free Full Text]
  18. Unfer RC, Hellrung D, Link CJ, Jr. 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 therapy. Cancer Res 2003;63:987–93.[Abstract/Free Full Text]
  19. LaTemple DC, Abrams JT, Zhang SY, Galili U. Increased immunogenicity of tumor vaccines complexed with anti-Gal: studies in knockout mice for {alpha}1,3galactosyltransferase. Cancer Res 1999;59:3417–23.[Abstract/Free Full Text]
  20. Gorelik E, Duty L, Anaraki F, Galili U. Alterations of cell surface carbohydrates and inhibition of metastatic property of murine melanomas by {alpha} 1,3 galactosyltransferase gene transfection. Cancer Res 1995;55:4168–73.[Abstract/Free Full Text]
  21. Rosenberg SA. Principles and practice of the biologic therapy of cancer, 3rd ed. PA: Lippincott Williams & Wilkins; 2000.
  22. Link CJ, Hellrung DJ, Seregina T, Wang S. Eliciting hyperacute rejection as a tumor killing strategy. Herpes amplicon vector transfer of the {alpha}(1,3)galactosyltransferase gene. Adv Exp Med Biol 2000;465:217–27.[Medline]
  23. LaTemple DC, Henion TR, Anaraki F, Galili U. Synthesis of {alpha}-galactosyl epitopes by recombinant {alpha}1,3galactosyl transferase for opsonization of human tumor cell vaccines by anti-galactose. Cancer Res 1996;56:3069–74.[Abstract/Free Full Text]
  24. Schuurhuis DH, Ioan-Facsinay A, Nagelkerken B, et al. Antigen-antibody immune complexes empower dendritic cells to efficiently prime specific CD8+ CTL responses in vivo. J Immunol 2002;168:2240–6.[Abstract/Free Full Text]
  25. Heath WR, Carbone FR. Cross-presentation, dendritic cells, tolerance and immunity. Annu Rev Immunol 2001;19:47–64.[CrossRef][Medline]
  26. Matzinger P. Tolerance, danger, and the extended family. Annu Rev Immunol 1994;12:991–1045.[Medline]
  27. Matzinger P. The danger model: a renewed sense of self. Science 2002;296:301–5.[Abstract/Free Full Text]
  28. Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol 2003;21:335–76.[CrossRef][Medline]
  29. Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2001;2:675–80.[CrossRef][Medline]
  30. Stager S, Alexander J, Kirby AC, et al. Natural antibodies and complement are endogenous adjuvants for vaccine-induced CD8+ T-cell responses. Nat Med 2003;9:1287–92.[CrossRef][Medline]
  31. Honey K, Rudensky AY. Lysosomal cysteine proteases regulate antigen presentation. Nat Rev Immunol 2003;3:472–82.[CrossRef][Medline]
  32. Li M, Davey GM, Sutherland RM, et al. Cell-associated ovalbumin is cross-presented much more efficiently than soluble ovalbumin in vivo. J Immunol 2001;166:6099–103.[Abstract/Free Full Text]
  33. Rafiq K, Bergtold A, Clynes R. Immune complex-mediated antigen presentation induces tumor immunity. J Clin Invest 2002;110:71–9.[CrossRef][Medline]



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