Role of α‐gal epitope/anti‐Gal antibody reaction in immunotherapy and its clinical application in pancreatic cancer

Abstract

Pancreatic cancer is one of the most common causes of death from cancer. Despite the availability of various treatment modalities, such as surgery, chemotherapy and radiotherapy, the 5‐year survival remains poor. Although gemcitabine‐based chemotherapy is typically offered as the standard care, most patients do not survive longer than 6 months. Therefore, new therapeutic approaches are needed. The α‐gal epitope (Galα1‐3Galβ1‐4GlcNAc‐R) is abundantly synthesized from glycoproteins and glycolipids in non‐primate mammals and New World monkeys, but is absent in humans, apes and Old World monkeys. Instead, they produce anti‐Gal antibody (Ab) (forming approximately 1% of circulating immunoglobulins), which specifically interacts with α‐gal epitopes. Anti‐Gal Ab can be exploited in cancer immunotherapy as vaccines that target antigen‐presenting cells (APC) to increase their immunogenicity. Tumor cells or tumor cell membranes from pancreatic cancer are processed to express α‐gal epitopes. Subsequent vaccination with such processed cell membranes results in in vivo opsonization by anti‐Gal IgG in cancer patients. The interaction of the Fc portion of the vaccine‐bound anti‐Gal with Fcγ receptors of APC induces effective uptake of the vaccinating tumor cell membranes by the APC, followed by effective transport of the vaccinating tumor membranes to the regional lymph nodes, and processing and presentation of the tumor‐associated antigens. Activation of tumor‐specific B and T cells could elicit an immune response that in some patients is potent enough to eradicate the residual cancer cells that remain after completion of standard therapy. This review addresses these topics and new avenues of clinical importance related to this unique antigen/antibody system (α‐gal epitope/anti‐Gal Ab) and advances in immunotherapy in pancreatic cancer.

Pancreatic cancer, which is the fifth leading cause of cancer death worldwide, is a devastating disease associated with an extremely poor prognosis.1 Immunotherapy designed to target tumor‐associated antigens (TAA) is a promising treatment approach for pancreatic cancer. However, immunotherapy alone is limited by the number of cytotoxic T lymphocytes (CTL) that can penetrate large pancreatic tumors. Therefore, there is a need to find other modalities of immunotherapy for the treatment of this disease. In part, this is due to the nature of pancreatic cancer, which is often highly immunosuppressive, diagnosed late, and with a short time to death.2 To develop efficient immunotherapy for pancreatic cancer, it is important to have an understanding of the following basic issues: (i) the nature of the immune network against autologous tumor cells; (ii) the identity of tumor antigens and means to evaluate the immune response in patients with pancreatic cancer; (iii) tumor escape mechanisms from the immune system and strategies to overcome them; and (iv) development of efficient immune interventions to eliminate pancreatic cancer cells. In particular, identification of appropriate tumor antigens is an essential step for the development of effective immunotherapy against pancreatic cancer.

Anti‐Gal is an IgG antibody (Ab) present in large amounts in normal subjects and patients with malignancies. Galili3 and Macher and Galili4 estimated that approximately 1% of B cells in humans can produce anti‐Gal. Most of these B cells (designated here as “anti‐Gal B cells”) are in a quiescent state within the lymph nodes and spleen. Natural anti‐Gal Ab is produced primarily by anti‐Gal B cells present along the gastrointestinal tract, because of continuous stimulation by bacteria of the natural flora.3, 4 Anti‐Gal Ab specifically interacts with α‐gal epitopes (Galα1‐3Galβ1‐4GlcNAc‐R) on cell surface glycolipids and glycoproteins. Once anti‐Gal Ab binds to α‐gal epitopes on the cells, its Fc portion readily binds to Fcγ receptor (FcγR) III on dendritic cells and macrophages. This interaction induces effective phagocytosis of the anti‐Gal‐Ab‐opsonized cells by antigen‐presenting cells (APC).5, 6, 7 Pig xenografts transplanted into humans release into the circulation α‐gal epitopes on xenoglycoproteins that reach the quiescent anti‐Gal B cells, activate them and induce extensive production of high affinity anti‐Gal IgG molecules.3, 4 Indeed, anti‐Gal Ab can bind extensively to α‐gal epitopes on xenograft cells and exacerbate xenograft destruction, primarily through antibody‐dependent cell‐mediated cytotoxicity (ADCC).3, 4 Whereas anti‐Gal Ab has a detrimental effect in xenotransplantation, its strong immunological potential might be exploited for immunotherapy in patients with pancreatic cancer. In previous studies, Rossi et al. exploited this unique immune mechanism to create a whole cell cancer vaccine to treat melanoma tumors.8, 9 B16 melanoma vaccines genetically modified to express α‐gal epitopes effectively targeted pre‐existing subcutaneous and pulmonary α‐gal‐negative melanoma tumors in α1,3‐galactosyltransferase (α1,3 GT) knockout mice.8, 9

In this article, we address the basic problem of cancer immunotherapy and detail our recent work in tumor antigen vaccination using α‐gal epitopes/anti‐Gal interaction. A series of preclinical experiments demonstrated that tumor‐associated antigens engineered to express α‐gal epitopes elicit enhanced immunogenicity and an effective antitumor immune response. We also discuss the novel approach of immunotherapy that targets pancreatic cancer stem cells using stem cell markers, remodeled to express α‐gal epitopes.

Distribution of α1,3 GT, α‐Gal Epitopes and Anti‐Gal Antibody in Mammals

The α‐gal epitope (Galα1‐3Galβ1‐4GlcNAc‐R) with a unique carbohydrate structure is absent in humans but naturally expressed on glycolipids and glycoproteins in non‐primate mammals, prosimans and New World monkeys.3, 4 In 1968, Yamakawa and colleagues were the first to isolate ceramidepentahexoside (CPH), a glycolipid, from rabbit red blood cells, which contained the non‐reducing terminal sequence, Galα1‐3Galβ1‐4GlcNAc‐R.10 Subsequently, the structure of the rabbit red blood cell CPH was further characterized by Hakomori and colleagues in 1973.11 The expression of α‐gal epitopes was assessed by measuring the binding of anti‐Gal Ab, a natural antibody, to this epitope in humans and to the α‐gal epitope‐specific lectin, Bandeiaea (Giffonia) simplicifolia IB412 in various cells. Although this epitope is not expressed on the cells of non‐mammalian vertebrates, it is abundantly expressed on cells of non‐primate mammals, including marsupials.3, 4 Among primates, the α‐gal epitope is found on red blood cells and nuclear cells of prosimans (e.g. lemura) and New World monkeys (e.g. monkeys of South and Central America). In contrast, Old World monkeys (e.g. monkeys of Asia and Africa), apes and humans lack α‐gal epitopes and instead produce large amounts of anti‐Gal Ab (Fig. 1).3, 4

Figure 1.

Reciprocal evolution of α1,3‐galactosyltransferase (α1,3 GT) enzyme activity, α‐gal epitopes and anti‐Gal antibody in mammals. α‐gal epitopes have been synthesized in mammals by α1,3 GT for more than 125 million years, since before the divergence of placental mammals and marsupials. All non‐mammalian vertebrates lack α1,3 GT and do not express α‐gal epitopes. Expression of this epitope was suppressed in ancestral Old World primates after they diverged from New World monkeys, and probably after apes and monkeys diverged from each other. Suppression of α‐gal epitopes was followed by production of anti‐Gal natural antibody, which is absent in non‐primate mammals, prosimians and New World monkeys.

The reason for this unique pattern of distribution is the differential activity of α1,3 GT, a glycosyltransferase enzyme.3, 4 This enzyme catalyzes within the Golgi apparatus the synthesis of α‐gal epitopes on the carbohydrate chains of glycolipids and glycoproteins. α1,3 GT appeared early in mammalian evolution, prior to the divergence of marsupials and placental mammals (>125 million years ago), and has been active in non‐primate mammals as well as in ancestral primates (Fig. 1).3, 4 However, after the geographic separation of South America from the African continent, ancestral pressure resulted in inactivation of the α1,3 GT gene. Cloning of the α1,3 GT gene in bovine and mouse cells enabled the identification of α1,3 GT pseudo‐genes in humans, Old World monkeys and apes.3, 4, 13, 14 Based on the sequences of pseudo α1,3 GT genes in various primates, this evolutionary inactivation is estimated to have occurred approximately 20–25 million years ago.3, 4, 13 A possible evolutionary scenario that could have resulted in inactivation of the α1,3 GT gene is the appearance of an infection agent that expressed α‐gal epitopes, which was detrimental to monkeys and apes, and endemic only in the Old World (i.e. it did not reach the South American continent due to geographic barriers). Such an infectious agent could have induced selective pressure for the evolutionary story of primates suppressing α‐gal epitope expression (i.e. inactivation of the α1,3 GT gene) and producing anti‐Gal Ab as a means of defense.3, 4

In 1984, Galili3 and Macher and Galili4 reported that 1% of circulating IgG in human sera showed specificity for α‐linked galactose. This antibody, anti‐Gal Ab, is found in high titer in sera of non‐immunocompromised humans. It is produced throughout life as a result of continuous antigen stimulation by carbohydrate antigens of the normal flora of the gastrointestinal tract, including Klebsiella pneumonia, Escherichia coli and Serratiamarcecens.3, 4 Characterization of the immunoglobulin genes encoding the anti‐Gal heavy chain indicates that a number of closely related genes encode this chain, implying that anti‐Gal Ab is a polyclonal antibody.3, 4 The polyclonality of anti‐Gal Ab was subsequently confirmed by isoelectric focusing analysis.3, 4 In spite of its polyclonality, anti‐Gal Ab interacts specifically with α‐gal epitopes on glycolipids and glycoproteins. The reason for this high specificity is that α‐gal epitope, like most other carbohydrate chains lacking sialic acid, is devoid of electrostatic charges. Thus, binding of anti‐Gal Ab to α‐gal epitopes is facilitated only by hydrogen bonds, hydrophobic interactions and van der Waal's forces.

Interaction of Anti‐Gal Ab/α‐gal Epitope is a Major Obstacle in Clinical Xenotransplantation

Transplantation of pig organs into humans, that is, xenotransplantaion, is a research field of considerable clinical significance since the number of human organ donors is insufficient.15, 16 Pigs are considered suitable organ donors because their organs are similar in size and function to many human organs.15, 16 However, the interaction of human natural anti‐Gal Ab with millions of α‐gal epitopes expressed on the pig cell surface causes strong xenograft rejection.3 The immunorejection is the result of cell lysis following complement activation by bound anti‐Gal Ab (complement‐dependent cytolysis) and ADCC, in which anti‐gal IgG binding to α‐gal epitopes on pig cells directs the subsequent binding of cytolytic effector cells, including macrophages and natural killer cells (Fig. 2).3 Pig kidneys and hearts transplanted into Old World monkeys, such as rhesus and baboon, are rejected immediately within 30 min to several hours by a process designated as hyperacute rejection (Fig. 2).3

Figure 2.

Mechanism of pig xenograft rejection. The interaction of human natural anti‐Gal with millions of α‐gal epitopes, expressed on the pig cell surface causes strong xenograft rejection. The in vivo binding of anti‐Gal antibody (Ab) to α‐gal epitopes on transplanted pig heart or kidney is the main cause of hyperacute rejection of such grafts in humans and in Old World monkeys. The recent generation of α1,3 GT‐knockout (KO) pigs that lack α‐gal epitopes has resulted in the elimination of this immunological barrier. α1,3 GT, α1,3‐galactosyltransferase; ADCC, antibody‐dependent cell‐mediated cytotoxicity; CDC, complement‐dependent cytolysis; ER, endoplasmic reticulum; FcγR, Fcγ receptor; NK, natural killer cells.

Extensive research has been performed to find methods that can prevent the hyperacute rejection. Because α1,3 GT competes enzymatically with other “masking” glycosyltransferases within the Golgi apparatus for the same accepter substrates, several research groups attempted to downregulate the expression of α‐gal epitopes by overinduction of α1,2fucosyltransferase17 or sialyltransferase18 in pig cells. However, these efforts did not completely eliminate the epitope, due to the fact that as many as 1% of human circulating B lymphocytes are quiescent but capable of anti‐Gal Ab production when activated.3, 4 The increase in the amount of high affinity anti‐Gal Ab is potent enough to mediate the killing of xenograft cells.3, 4

Epidemiology of Pancreatic Cancer and Clinical Management

Pancreatic cancer is highly aggressive and is the fifth leading cause of cancer death in Japan with over 27 000 estimated new cases in 2009.22, 36 The prognosis of these patients is dismal with overall survival <5% and a median survival of 4–6 months. At the time of diagnosis, 15–26% of patients present with operable disease, whereas approximately 30% have locally advanced, unresectable disease and approximately 44% have metastatic disease. Surgical resection remains the only potentially curative intervention19 but is contraindicated in most patients because the disease is diagnosed at an advanced stage. However, even with complete surgical resection, recurrence is common and the majority of patients develop recurrence with distant metastases. The median survival time after surgery is 15–20 months with a 5‐year survival rate of approximately 20%.19, 20 The median survival time of patients with locally advanced, unresectable disease is 10–12 months.19, 21

The poor prognosis of pancreatic cancer is related to a combination of late detection, as most patients present with locally advanced or metastatic disease, and standards of care that consist of relatively ineffective chemotherapeutic regimens. Gemcitabine is currently approved and the chemotherapeutic agent of choice, for the treatment of patients with pancreatic cancer, with adjuvant chemotherapy now considered the standard of care in many postoperative pancreatic cancer patients treated in Japan.22 Several promising drugs that target the main aspects of malignancy, such as angiogenesis, proliferation and metastasis, have failed to provide clinically relevant benefits, despite trivial improvement in disease‐free survival and overall survival rates.23 Therefore, new therapeutic approaches need to be encouraged and investigated.

Immunotherapy is an innovate approach that uses techniques such as vaccination designed to activate the patient's immune system with TAA expressed in pancreatic cancer cells. While several clinical studies have documented evidence of treatment‐induced, antigen‐specific immune responses, few, if any, protective immune responses have been observed in patients with metastatic disease. Despite this setback, there is renewed optimism for immunotherapy, since vaccination after surgery in patients with no or minimal disease has been reported to have an impact in pancreatic cancer.24 In addition, vaccination against tumor antigens is an attractive approach to adjuvant treatment post‐surgery, when tumor‐induced immune suppression is minimal.25, 26

Targeting Whole‐Cell Vaccines to APC

The simplest vaccine approach that has been applied to cancer is inoculation of individuals with irradiated tumor cells. This approach has the following advantages: (i) specific tumor antigens do not need to be identified or characterized prior to vaccination; (ii) immune responses to multiple tumor antigens can be generated, which might protect against tumor escape variants;27 (iii) such vaccines are not limited by patient human leukocyte antigen (HLA) background due to cross‐presentation of tumor antigens after uptake by dendritic cells (DC),27, 28 which is particularly advantageous as tumor cell lines are readily available, while the availability of autologous tumor cells might be limited; and (iv) the tumor cell vaccine platform can be easily modified. For example, tumor cells can be transduced to express immunomodulatory cytokines such as granulocyte macrophage colony‐stimulatory factor (GM‐CSF), as reported by Jaffee and colleagues in a phase I clinical study.29 They tested a new vaccine in an adjuvant setting in patients with resected pancreatic cancer. Two cell lines of pancreatic cancer were tested, both of which were genetically modified to express the human GM‐CSF described above. The tumor cell vaccines were administrated to 14 patients who had undergone pancreaticoduodenectomy. Delayed‐type hypersensitivity in response to the autologous tumor cells occurred in three patients, who also had disease‐free survival of longer than 25 months at the time the study was conducted.29 These pancreatic tumor cell vaccines induce a CD8⁺ T‐cell response, specific mesothelin, regardless of the HLA match between the tumor vaccine and the recipient, demonstrating the occurrence of cross priming.28, 29 Mesothelin, a cell‐surface glycoprotein, is a particularly promising cancer vaccine target due to its low level of expression in healthy pancreatic tissue and high levels of expression in pancreatic as well as other cancers (e.g. ovarian).30 A phase II trial of these vaccines is currently being conducted in patients with resectable pancreatic cancer (NCT0038610). Unfortunately, TAA present in tumor cells cannot efficiently induce an antitumor immune response by themselves, because tumor cells lack costimulatory molecules.31, 32 Effective induction of an antitumor immune response by a tumor cell vaccine requires uptake of tumor cells or tumor cell membranes by professional APC, processing of TAA molecules, presentation of TAA antigenic peptide on APC in association with MHC class II molecules for activation of specific CD4⁺ helper T cells, and association with MHC class I molecule for activation of CD8⁺ cytotoxic T cells (CD8⁺ CTL) (first signal).31, 32 In addition, activation of tumor‐specific T cells requires the delivery of a costimulatory non‐specific signal (second signal), which can be provided by interaction between costimulatory molecules expressed on the membrane of APC and T cells. Accordingly, T cells require two signals to become fully activated.32 Another reason for the absolute need for effective uptake of tumor cell vaccine by APC is that activation of TAA‐specific T cells does not occur at the vaccination site, but rather within the draining lymph nodes of the vaccination site or within the spleen. Only after they are activated can tumor‐specific T cells leave the lymph nodes or spleen to seek and destroy tumor cells expressing the TAA. For such activation to occur, the tumor vaccine must be transported from the vaccination site by APC to the lymph nodes or to the spleen.33

In Vivo Targeting of Tumor Cell Vaccine to APC by α‐Gal Epitopes/Anti‐Gal Interaction

As described in the previous sections, TAA molecules on pancreatic cancer cells do not express markers that contain modification of TAA to be recognized by APC. To increase the immunogenicity of TAA against APC, IgG bound to TAA could be a suitable strategy. The APC, including macrophages, skin Langerhans cells and blood‐derived DC, all express FcγR (e.g. Fcγ RI/CD64, Fcγ RII/CD32, Fcγ RIII/CD16). These FcγR can effectively bind and mediate the internalization of opsonized cells (i.e. cells with bound IgG molecules), cell membranes or molecules (all defined as cancer antigen) via the Fc portion of the opsonizing IgG antibody.34, 35 This results in effective enhancement of the immunogenicity of the antigen complexed with an IgG antibody. Thus, vaccination of cancer patients with a tumor cell vaccine, modified to express α‐gal epitopes, should result in in situ binding of the patient's anti‐Gal IgG molecules to α‐gal epitopes on the vaccinating cell membrane. This targets the vaccines to APC by interaction of the Fc portion of anti‐Gal Ab on the vaccinating cell membrane with FcγR on the APC.6, 7 This interaction induces the uptake of the vaccine by APC, which subsequently transport the vaccinating tumor membranes to the draining lymph nodes. Once the TAA‐specific T cells are activated, they can leave the lymph node, circulate in the body and seek malignant cells expressing the TAA in order to destroy them. There is an ongoing clinical immunotherapy study, which is being conducted as a clinical phase III study in patients with surgically resected pancreatic cancer (NCT01072981). The purpose of the study is to assess overall survival after treatment with a regimen of adjuvant therapy (Gemcitabine alone or with 5‐fluorouracil [5‐FU] chemoradiation) with or without experimental immunotherapy using HyperAcute Pancreas (Algenpantucel‐L), which consists of two separate allogeneic cancer cell lines engineered to express α‐gal epitopes in subjects who have undergone surgical resection. At present, this study is currently recruiting participants until January 2014, the estimated study completion date.

Immunotherapy with MUC1‐Based Vaccine for Pancreatic Cancer

In our previous study, we investigated the effects of vaccination with α‐gal epitope‐expressing pancreatic cancer cells and examined the usefulness of this vaccine in the induction of tumor‐specific T‐cell responses, in vivo prevention of tumor growth and improvement of survival.36 We used a human pancreatic cancer cell line, PANC1, which endogenously expresses Mucin 1 (MUC1) protein in the tumor cell vaccine. MUC1 is the most notable tumor antigen of pancreatic cancer. This molecule is a large membrane glycoprotein that consists of multiple 20 amino acid repeats, which are heavily O‐glycosylated. Expression of the glycosylated MUC1 is normally restricted to the apical surface of epithelial ducts. Both the glycosylation pattern and surface expression pattern change dramatically in cancer tissues. Tumor MUC1 is underglycosylated and no longer restricted to a particular surface of the cell as tumor cells lose polarity.37, 38 This change in the expression profile of tumor MUC1 leads to increased processing and presentation of the protein backbone to the immune system. Numerous studies reported identical anti‐MUC1 immune responses in cancer patients. The eventual goal of MUC1‐based immunotherapy is to enhance these immune responses with the hope of facilitating tumor rejection. In addition, a MUC1‐based vaccine could be useful prophylactically in high‐risk individuals.39 In fact, several clinical trials of MUC1‐based vaccines have already been tested.2, 40 However, the vaccine has not been as successful as hoped, because MUC1 proteins on pancreatic cancer cells do not express markers that contain modification of MUC1 to be recognized by APC. To increase the immunogenicity of both PANC1 tumor cell vaccine and MUC1 antigen against APC, we modified these cells to express α‐gal epitopes by mouse α1,3 GT gene transfection (designated here as α‐gal PANC1) (Fig. 3). This modified tumor cell vaccine takes advantage of anti‐Gal Ab found in most people based on exposure to gastrointestinal flora, resulting in an increased uptake of the vaccine in an antibody‐dependent manner. Moreover, MUC1 can also be remodeled to express α‐gal epitopes, because the MUC1 molecule has five potential sites of N‐glycans and can bind anti‐Gal in situ at the vaccination site (Fig. 3). Using mice in our experimental studies, we investigated the effects of α‐gal PANC1 vaccine in eliciting both antibody production (Fig. 4a) and T cell responses (Fig. 4b) against MUC1 antigen. The results showed that α‐gal PANC1 vaccines protected and prolonged the survival of α1,3 GT knockout mice harboring tumors of MUC1‐B16F10 melanoma cells that had been sensitized by pig kidney fragment (Fig. 5).36

Figure 3.

Increased immunogenicity of tumor‐associated antigen (TAA), MUC1, engineered to express α‐gal epitopes. Immunity towards TAA, including MUC1, in cancer patients is relatively weak and presentation of these TAA to the immune system is poor due to low immunogenicity. We tested the effects of vaccination using immunogenetically enhanced MUC1 (by expressing α‐gal epitopes) on production of antibodies for MUC1 as well as other TAA derived from pancreatic cancer cells, and induction of tumor‐specific T cell responses. α1,3 GT, α1,3‐galactosyltransferase; APC, antigen‐presenting cells; CTL, cytotoxic T lymphocytes; FcγR, Fcγ receptor.

Figure 4.

Expansion of B and T cells in response to cell vaccination. (a) ELISPOT assay for anti‐MUC1 antibody (Ab)‐producing B cells with lymphocytes from α‐gal PANC1‐vaccinated knockout (KO) mice and parental PANC1‐vaccinated KO mice. Parental PANC1‐vaccinated KO mice had 320 ± 19 spots/10⁶ splenocytes compared with 554 ± 67 spots/10⁶ splenocytes in α‐gal PANC1‐vaccinated KO mice. The latter included a significantly higher proportion of anti‐MUC1 B cells (P < 0.0001). (b) ELISPOT assay for in vitro activated T cells detected using interferon‐γ secretion. In parental PANC1‐vaccinated KO mice, 211 ± 33.4 and 153 ± 15.2 spots/10⁶ splenocytes were detected in the presence or absence of MUC1 peptide, respectively, and no significant difference in the number of spots was observed. In contrast, α‐gal PANC1‐vaccinated KO mice displayed 1238 ± 283 spots/10⁶ splenocytes in the presence of MUC1 peptides, but only 314 ± 49 spots/10⁶ splenocytes in the absence of MUC1 peptides.

Figure 5.

Photographs of growing tumors in vaccinated knockout (KO) mice after tumor cell challenge with MUC1‐B16F10 cells and resultant in vivo prevention of tumor growth and prolongation of survival. (a) Representative photographs of mice treated with either α‐gal PANC1 or parental PANC1 vaccines. The tumor size in each mouse treated with α‐gal PANC1 vaccine was smaller than that of mice treated with parental PANC1 vaccine. (b) Tumor size of mice vaccinated with parental PANC1 doubled every 3–7 days and reached a maximum size of ~980 mm² within 22–25 days. Tumors of mice treated with α‐gal PANC1 continued to grow in non‐vaccinated α1,3‐galactosyltransferase KO mice but displayed a much slower growth rate than most parental PANC1‐treated KO mice. (c) The mean survival time of KO mice treated with α‐gal PANC1 was significantly longer (41.4 ± 10.5 days) than that of KO mice treated with parental PANC1 vaccine (21.1 ± 10.5 days, P = 0.003).

α‐gal PANC1 Vaccine Elicits an Immune Response Against Pancreatic Cancer Stem Cells

A rare population of cells with stem cell properties called cancer stem cells (CSC) was identified recently.41 The CSC are generally dormant or slowly cycling tumor cells that can reconstitute tumors. Based on this new finding, it is necessary to develop effective therapies for CSC to achieve complete eradication of cancer cells. Apart from reconstituting tumor cells, CSC are also important because they are considered to be involved in resistance to chemotherapy and radiotherapy, tumor relapse and progression. In 2007, Li and colleagues reported that putative pancreatic CSC express CD44, CD24 and epithelial‐specific antigen (ESA), which are glycoprotein molecules, and that they can be used as markers to identify CSC.42 Accordingly, we hypothesized that biosynthesis of α‐gal epitopes on the carbohydrate of CSC markers expressed on pancreatic CSC could effectively induce antibody production against these stem cells. In our previous study, sera obtained from α‐gal PANC1‐vaccinated KO mice produced anti‐CD44⁺ CD24⁺ PANC1 antibody, but those from parental PANC1‐vaccinated KO mice were negative for anti‐CD44⁺ CD24⁺ PANC1 antibody (Fig. 6). These findings suggest that the build‐up of α‐gal epitopes on the carbohydrate of CSC markers, thus allowing those molecules to be internalized by APC, is a potentially useful strategy to eliminate cancer cells, including differentiated cancer cells and pancreatic CSC, and might open a window of opportunity for cure of pancreatic cancer by the destruction of micro‐metastasis and minimal residual disease. Various CSC markers, including CD44, CD24, ESA and CD133, were used previously to identify CSC in pancreatic cancer and other type of cancers.43, 44, 45 As shown in Table 1, these CSC markers are glycoproteins and α‐gal epitopes could be biosynthesized on the carbohydrate of these CSC markers. Therefore, our novel immunotherapy, using α‐gal epitopes/anti‐Gal interaction, can be applied to not only pancreatic cancer but also to other types of cancer, including colorectal, ovarian and gastric cancer.

Figure 6.

Production of antibodies against cancer stem cells assessed using flow cytometry assay. (a) Production of anti‐PANC1 antibody (Ab) in sera of vaccinated knockout (KO) mice. (b) Production of anti‐CD44⁺ CD24⁺ PANC1 (i.e. pancreatic cancer stem cells) Ab in sera of vaccinated KO mice. (c) Production of anti‐CD44⁻ CD24⁻ PANC1 (i.e. differentiated pancreatic cancer cells) Ab in sera of vaccinated KO mice. (a–c) Closed histogram, unstained cells; open histogram, stained cells with sera from vaccinated KO mice.

Table 1.

Markers of cancer stem cells (CSC) in various cancers

Cancer	CSC marker
Pancreatic cancer	CD44, CD24, ESA, CD133
Gastric cancer	CD44, CD133
Colorectal cancer	CD44, CD133, CD166, CD326 (EpCAM)
Hepatocellular carcinoma	CD90
Glioblastoma	CD133
Head and neck cancer	CD44
Ovarian cancer	CD133
Malignant mesothelioma	CD24, CD26

All stem cell markers described in Table 1 are glycoproteins.

EpCAM, epithelial cell‐specific adhesion molecule; ESA, epithelial‐specific antigen.

Although MUC1 protein is a potential target in immunotherapy for pancreatic cancer, vaccination against a single TAA is disadvantageous because it is not clear which antigens, including MUC1, can potentially induce effective antitumor immune responses. Furthermore, immunity against various antigens is expected to be more effective in heterogeneous cell populations of cancers compared with a single antigen. In our study, we used a vaccine of whole cancer cells, which upregulated the immunogenicity of well characterized as well as uncharacterized multiple TAA contained in cancer cells by biosynthesis of α‐gal epitopes on the carbohydrate of TAA. As shown in Table 2, several TAA of pancreatic cancer, including MUC1 and mesothelin are glycoproteins and thus the use of a vaccine of whole cancer cells could effectively induce APC to internalize these TAA. Multiple TAA can be presented to T cells by both MHC class I and class II pathways, ultimately leading to polyclonal expansion of both B and T cells.

Table 2.

Overview of candidate pancreatic cancer‐associated antigens for immune targeting

Antigen	Location	Molecular type	Expression in tumor	Prevalence (%)
Mesothelin	Cell surface (GPI linked)	Glycoprotein	Overexpressed	90–100
CEA	Cell surface (GPI linked)	Oncofetal protein	Overexpressed	30–100
Her2‐neu	Transmembrane	Oncoprotein	Overexpressed	>50
K‐Ras	Intracellular	Oncoprotein	Mutated self	90
MUC1	Transmembrane	Glycoprotein	Overexpressed hypoglycosylation	90
p53	Intracellular	Tumor‐suppressor protein	Mutated self	50–70

CEA, carcinoembryonic antigen; GPI, glycosylphosphatidylinositol; Her2‐neu, human epidermal growth factor receptor 2; K‐Ras, v‐Ki‐ras2 Kirsten rat sarcoma viral oncogene homolog.

To further develop effective immunotherapy of pancreatic cancer, we proposed the use of tumor cell lysate as the source of tumor antigens, because such lysate contains several known as well as unknown antigens that can elicit an antitumor immune response. In other words, vaccines prepared from tumor cell lysates can provide both multiple TAA of differentiated cancer cells and stem cell markers of CSC. However, it is noteworthy that induction of the immune response against CSC by standard vaccination with tumor cell lysate is often difficult, because the proportion of CSC is only 1% of all cancer cells. Accordingly, we plan to generate a vaccine prepared from tumor lysate that is enzymatically engineered to express α‐gal epitopes. It is hoped that such a vaccine can elicit an effective immune response toward both TAA and CSC markers to eliminate the replenishing pool of all pancreatic cancer cells.

Closing Remarks

The reason for the lethal nature of pancreatic cancer is the ability of remnant tumor cells, including differentiated cancer cells and CSC after surgery, chemotherapy and radiation therapy, to develop into a recurrent or metastatic tumor. These remnant residual cancer cells cannot be detected by imaging, but their destruction might be achieved by activation of immunocytes that can specifically attack and destroy TAA‐expressing tumor cells. Microarray technology and serial analysis of gene expression have identified new targets for vaccine development. However, advances in biotechnology are also needed for vaccine administration and for eliciting the most effective immune response. The most encouraging results of immunotherapy in pancreatic cancer have been seen in adjuvant settings, such as post‐surgery. Moreover, immunotherapy studies on specific TAA have identified the inefficiency of mono‐TAA‐based immunization in eliciting a clinically protective immune response. Due to genome instability, the immunological selection pressure for the destruction of TAA‐expressing tumor cells frequently results in the appearance and expansion of tumor cell subclones with no or low expression of the specific TAA.46, 47, 48 Therefore, any vaccine used for patients with pancreatic cancer should contain multiple TAA, that is, a polyvalent tumor vaccine, prepared from autologous tumors. Recent publications have demonstrated the presence of multiple mutations in pancreatic cancer that can be targeted, that many of these mutations are already present in primary pancreatic cancers and that vaccination after resection of the primary tumor might protect against recurrence.49, 50 These findings suggest that personalized vaccines tailored toward the mutations found in individual patients might be necessary for successful immunotherapy.51 Exploiting anti‐Gal as an antibody targeting vaccine for APC can overcome the inefficient autologous tumor vaccine uptake by APC, provided that the vaccine expresses α‐gal epitopes.

To establish this novel immunotherapy as the next generation therapeutic option in pancreatic cancer, clinical studies involving the application of immunotherapy using tumor lysate, remodeled to express α‐gal epitopes, should be conducted.

Disclosure Statement

The authors have no conflict of interest and the present study does not include discussion of off‐label or investigational use.

(Cancer Sci, doi: 10.1111/cas.12084, 2013)