MUC1 immunotherapy - Future Medicine

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MUC1 immunotherapy The overexpression and aberrant glycosylation of MUC1 is associated with a wide variety of cancers, making it an ideal target for immunotherapeutic strategies. This review highlights the main avenues of research in this field, focusing on adenocarcinomas, from the preclinical to clinical; the problems and possible solutions associated with each approach; and speculates on the direction of MUC1 immunotherapeutic research over the next 5–10 years. KEYWORDS: aberrant O-linked glycosylation cancer episialin immunotherapy MUC1 polymorphic epithelial mucin

Immunotherapy is based on a simple premise: to manipulate the natural immunological mechanisms for a present or future clinical benefit. In order to utilize these underlying mechanisms effectively, we must first understand them and, as such, it is not a discipline where the advancement of scientific knowledge will necessarily have an immediate impact in the clinic. Despite significant progress being made in our understanding of T-cell-mediated antitumor responses and the immense inroads that have been made into our understanding of basic immunology, the response rate for immunotherapy for solid tumors is very low. Indeed, in a review conducted in 2004 of 1306 cancer vaccine treatments, an objective response rate of only 3.3% was seen [1] . However, we are now in a period where our understanding is beginning to allow us to play a more active therapeutic role, and the genetic revolution, giving us highly controllable systems, is now part of the mainstream of immunotherapy. The aim of this review is to discuss the recent advances in MUC1 immunotherapy focusing on adenocarcinomas, drawing on past lessons and speculating on how the field will develop over the next 5–10 years.

MUC1 In 1999, it was estimated that cancers demonstrating an upregulation of MUC1 accounted for approximately 72% of new cases and for 66% of deaths in all cancers [2] . This is an astonishing fact, which immediately brings into focus Ehrlich’s magic bullet hypothesis; that we may have found a near universal marker to target malignant cells. However, as this review will discuss, despite a prolonged period of

investigation – MUC1’s link with cancer was first observed in 1981 [3] – therapies using MUC1 as the target are not in clinical use, although several Phase III clinical trials are ongoing [301] . MUC1 (for other names, see TABLE 1), which is encoded on chromosome 1q21 [4] , is a large [5] epithelial glycoprotein of the mucin family, of which there are currently known to be 21 members. Mucins are defined as glycoproteins carrying greater than 50% of their mass in O-linked glycans, but otherwise differ with regard to their structure and function. Mucins can be broadly split into two groups: gel-forming and membrane-bound. MUC1 is a membranebound mucin, but the extracellular domain can be cleaved and released in both cancerous and noncancerous scenarios [6–9] . Like all mucins, MUC1 has a ‘tandem repeat’ amino acid sequence and a high percentage of carbohydrates (50–90% of mass), predominantly O-linked to serine or threonine residues [5,10] . Each tandem repeat of MUC1 is 20 amino acids long with five potential O-linked glycosylation sites (PDTRPAPGSTAPPAHGVTSA) and the number of these repeats is allele dependent, varying from 20 to 125 [11–13] , hence the widely used abbreviation ‘VNTR’ for variable number of tandem repeats. This region now appears to be less uniform than originally thought, since polymorphisms have been observed in several studies and this may have significance with regard to MUC1’s immunogenicity [14,15] . One alteration that has been seen in all individuals assessed is the change from PDTR to PESR, with variations in its frequency and location within the repeat region. Three other variations result in the substitution of a specific proline (TAPPA) with Q, A or T, but these are less common. It is important

10.2217/IMT.10.17 © 2010 Future Medicine Ltd

Immunotherapy (2010) 2(3), 305–327

Richard E Beatson1, Joyce Taylor-‐ Papadimitriou1 & Joy M Burchell†1 1

Breast Cancer Biology Group, King’s College London, Guy’s Hospital, London SE1 9RT, UK † Author for correspondence: Tel.: +44 020 7188 1470 Fax: +44 020 7188 0919 [email protected]

ISSN 1750-743X

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Beatson, Taylor-Papadimitriou & Burchell

Table 1. MUC1 nomenclature. Acronym

Full name

PubMed hits (search performed in November 2009)

Year first used

Points to note

MUC1

Mucin 1

4267

1979

PEM

Polymorphic epithelial mucin

3950

1979

Refers to all species, all isoforms and all glycoforms Note: MUC1 = human, Muc1 = murine Refers to all species, all isoforms and all glycoforms

PEM

Episialin

3906

1979

Refers to all species, all isoforms and all glycoforms

MSA

Mammary serum antigen

676

1964

CA15–3

Cancer antigen 15–3

333

1986

Refers to all species and all glycoforms, but only serum MUC1 Human serum MUC1

KL6

(Antibody clone)

318

1988

DF3

(Antibody clone)

91

1984

27

1984

MAM-6

Also the name of the antibody Human sialylated serum MUC1 Also the name of the antibody Surface-bound human MUC1 Human MUC1

PUM

Peanut-reactive urinary mucin 9

1983

CD227

Cluster of differentiation 227

7

2001

PAS-0

4

1982

Human MUC1 that reacts with peanut agglutinin lectin (terminal galactose, but not GalNAc) Refers to human MUC1, all isoforms and all glycoforms Human MUC1

CAM 123–6

3

1994

Human serum MUC1

to note that the glycosylation sites and proline residues, both so important for function, appear to be largely preserved. MUC1’s glycosylation is dense and this density is crucial for its function, giving MUC1 wideranging binding and water-holding properties, as well as insensitivity to proteases and the ability to bind to pathogens [6,7,16] . This dense glycosylation, allied to a large number of proline residues, gives MUC1 a ‘bottlebrush-like’ structure, maintaining a linear form and allowing it to extend approximately 200–500 nm from the cell surface – far further than the glycocalyx (10 nm) and surface receptors [17,18] . MUC1 is transcribed as one, before being cleaved in the endoplasmic reticulum (ER) into two subunits (D and E), which are held together on the cell surface in a heterodimeric complex by strong noncovalent linkages [10] . MUC1 is ubiquitous, being found throughout the body on the luminal surface of most simple epithelial cells, including the breast luminal epithelial cells [19] . Lower levels of MUC1 can also be observed on leukocytes [20–22] , erythroid cells [23] and, as has recently been demonstrated, on cord stem cells [24] .

MUC1 as an immunotherapeutic target  Characteristics of MUC1 that make it a potential target Tumor-associated antigens (TA As) can be broadly split into seven groups [25] : 306

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 Cancer germ-line genes (e.g., MAGE);  Novel antigens encoded by tumor-specific transcripts (e.g. TRP2);  Viral antigens (e.g., Epstein–Barr virus);  Antigens resulting from altered post-translational modifications (e.g., MUC1);  Antigens resulting directly from genetic mutation, deletions and substitutions (e.g., Bcr-abl);  Differentiation antigens (e.g., CEA);  Overexpressed tumor antigens (e.g., HER2 and MUC1). As can be seen, MUC1 sits in two such categories: overexpression and altered post-translational modifications and, as such, it is a desirable target. The expression of MUC1 is upregulated in most adenocarcinomas (FIGURE 1) and by more than 90% of breast carcinomas [13,26,27] . These expression levels increase by up to 100-fold as determined by RNA expression [5] and, in addition, there can be dramatic changes in O-linked glycosylation. These changes can easily be seen using immunohistochemistry (FIGURE 2) . It is also pertinent to note that MUC1 overexpression and aberrant glycosylation is not only associated with adenocarcinomas – it has also been found in squamous cell carcinomas [28–31] and myelomas [32] . Glycosylation is determined by the expression, activity and localization of specific glycosyltransferases. Different tissues can express future science group

MUC1 immunotherapy

90%

60%

90%

Breast

Lung

Ovarian

Metastatic prostate

70%

80%

90%

70%

Colorectal

Stomach

Pancreas

Kidney

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60%

Figure 1. MUC1 is overexpressed in many adenocarcinomas. Diagrammatic representation of the expression of MUC1 in various cancers. Adapted from a figure that was kindly supplied by Nadine Bizouarne from Transgene.

different glycosyltransferases and so the glycosylation of MUC1 will be dependent on the tissue in which it is expressed [13,33] . As a consequence, the changes in the glycosylation observed on MUC1 when expressed by malignant cells will reflect the tissue of origin of the carcinoma. To illustrate this, the glycosylation of MUC1 expressed by the normal mammary gland and the changes that occur in breast cancer will be described (F IGU R E 3) . In the

Normal MUC1 epithelial cells

normal mammary gland, the O-linked glycans carried on MUC1 are elongated and based on a core-2 branch [34] . This branch is initiated by the action of the glycosyltransferase, C2GnT1. This enzyme uses GalE1,3GalNAc as a substrate, which is known as core-1 and also forms the substrate for the competing sialyltransferases ST3Gal-I [35] (C2GnT1 cannot act when Neu5Ac is present on the galactose) and ST6GalNAc-II. Thus, the relative expression of these glycosyltransferases determines

Malignant MUC1 epithelial cells

Figure 2. Binding of HMFG2 to MUC1 in breast carcinoma. Primary invasive ductal breast carcinoma stained with HMFG2, a monoclonal antibody that preferentially reacts with MUC1 expressed by tumor cells. Adjacent normal tissue allows the differential reactivity of HMFG2 to be seen.

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Healthy

Carcinoma Core-2 β1,3 β1,6

Ser/Thr

MUC1-Tn

ST6GalNAc-I Ser/Thr

α2,6

Ser/Thr

MUC1-STn

Ser/Thr

MUC1-T

Ser/Thr

MUC1-ST

C2GnT-I

Core-1 β1,3

Branched polylactosamine chains

ST3Gal-I

α2,3

β1,3

Terminal glycan ST6GalNAc-II

α2,3

β1,3

Ser/Thr

MUC1-DiST

Key

-2

Co

re

re -1

Co C2GnT-I

GalNAc (N-acetylgalactosamine)

O-linked glycans added to MUC1 – catalyzed by glycosyltransferases

GlcNAc (N-acetylglucosamine)

C2GnT-I

Galactose

ST3Gal-I

ST3Gal-I

Golgi

Neu5Ac (sialic acid)

ST6GalN Ac-I Immunotherapy © Future Science Group (2010)

Figure 3. MUC1 is aberrantly glycosylated in breast cancer. On the left, in the healthy scenario, MUC1 is O-glycosylated with long core-2 glycans. The glycosyltransferase C2GnT1 initiates the core-2 branch. On the right, in the cancerous scenario, MUC1 carries truncated O-linked glycans, the most common forms being MUC1-Tn, MUC1-STn, MUC1-T, MUC1-ST and MUC1-DiST. See text for details of the mechanism. MUC1-STn is present on approximately 25% of breast cancers and core-1 structures are displayed on approximately 90% of breast cancers. However, it is important to remember that the glycan expression is not homogeneous. STn: Sialyl Tn.

whether glycoproteins with O-linked glycans carry core-2 glycans that allow further extension, or core-1 glycans that result in chain termination [13,36,37] . Breast cancers contain truncated O-linked glycans and this has been demonstrated to be due to the upregulation of the sialyltransferase ST3Gal-I [38] and the turning on of transcription of the sialyltransferase ST6GalNAc-I [39–41] . As these changes are commonly observed in breast cancer, it can be speculated that this may be advantageous for the tumor cell and so contribute to tumorigenesis. 308


In addition, during the change to malignancy, the location of the MUC1 glycoprotein is altered: cells lose their polarity, which results in MUC1 being expressed over the entire cell surface. Overexpression of MUC1 in depolarized cells has been demonstrated to decrease their adhesion to each other and the extracellular matrix, suggesting an increase in metastatic potential [42,43] .  Natural immune responses to cancer-associated MUC1 It appears that MUC1 expressing normal core-2 glycans is immunologically inert, although some future science group

MUC1 immunotherapy

cancer-associated MUC1 antibodies can bind with low affinity. Indeed, it has recently been demonstrated that 1–3% of medullary thymic epithelial cells, (cells involved in the induction of central tolerance) express core-2 MUC1 with no core-1 expression [44] . However, the cancerassociated change in the glycans carried by MUC1 exposes regions which can and do generate natural immune responses. Antibodies (IgG and IgM) to cancer-associated MUC1 are common and increased levels of these antibodies have been correlated with increased survival in breast cancer [45,46] and gastric cancer [47] ; however, a less clear correlation exists in colorectal cancers [48] . It is worth noting that the high number of IgM antibodies seen in so many studies are, when isolated or cloned, carbohydrate-specific [49] . This is almost certainly due to the ability of B cells to produce this class after carbohydrate-dependant crosslinking of B-cell receptors. This process is therefore independent of the antigen presenting cell (APC)–T-cell axis. Several studies have demonstrated the presence of MHC-restricted MUC1-specific cytotoxic T lymphocytes (CTLs) in pancreatic, ovarian and breast cancer patients [50–52] . Furthermore, it was found that MUC1-specific T cells in patients’ circulation are often anergic, yet T cells with the same specificity in the bone marrow were not [51,53] , emphasizing the importance of the tumor microenvironment. The potency of any response may also be influenced by underlying conditions that may prime the immune system through exposure to cancer-associated glycoforms of MUC1 [54–59] .  Function of MUC1 Numerous functions have been assigned to MUC1, ranging from its role as a physical barrier against pathogens [6,7] to its role in signaling through its 72-amino acid cytoplasmic tail (CT). The CT is highly conserved across species, confirming its functionality [60] , and can locate to the cytosol, mitochondria and nucleus, facilitating its interactions. A detailed description of the function of MUC1 is not within the scope of this review and readers are referred to reviews in this area [17,61] . However, it is important to note that recent data regarding MUC1 function suggest: that it may have an oncogenic effect [62–64] ; that tumor-associated glycoforms may interact with receptors of the immune system [65–67] ; that MUC1 may promote EGF-receptor signaling and vice versa [68,69] ; that MUC1 can interact directly and indirectly with p53 [70,71] ; that MUC1 can promote mammary tumorigenesis future science group

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in a murine model [72] ; and that MUC1 has a role in cell–cell interactions [5,18] . All these functions may have an influence on cancer progression and thus provide further data in support of the immunotherapeutic targeting of MUC1.

MUC1 immunotherapy TABLE 2 describes recent key clinical trials targeting MUC1. Passive immunotherapy  Monoclonal antibodies Monoclonal antibodies can be an extremely potent tool, with 21 being US FDA approved and used in clinics in 2008 [73] . The upregulation and change in glycosylation of MUC1 observed in cancer, coupled with its restriction to the luminal surface in normal cells (hence its poor accessibility in the normal scenario) indicate that antibodies directed against cancerassociated MUC1 could have a high degree of specificity for tumor cells. Many anticancer-associated MUC1 monoclonal antibodies were developed by immunizing mice with whole breast carcinoma tumor cells, transformed epithelial membrane lysates or fractions of breast milk. These antibodies were vital in deciphering the MUC1 amino acid sequence via expression cloning, and successfully stained primary lesions and distant metastases of many cancers. It was found that the VNTR was extremely immunogenic, especially the APDTR region [74] , and preclinical studies using some of these antibodies in murine models have been encouraging. In a spontaneous murine model of breast cancer, SM3, a monoclonal antibody that demonstrates a high specificity for tumorassociated MUC1 [27] was effective at delaying tumor growth [75] . PAM4 reacts with 85% of pancreatic adenocarcinomas, with no reactivity to healthy tissue. Combining PAM4, or I131-labeled PAM4, with chemotherapy to treat nude mice bearing human pancreatic tumors demonstrated a significant decrease in tumor growth, with the authors emphasizing that both treatments were required [76] . Bispecific antibodies anti-MUC1–anti-CD3 and anti-MUC1– anti-CD28 enhanced cytotoxity in vitro and inhibited the growth of bile duct carcinoma in SCID mice. The authors followed-up with an anti-MUC1–anti-CD2 antibody and found an improved result in the same model using all three bispecific antibodies [77] . However, despite the positive mouse data, there have been very few anti-MUC1 monoclonal clinical trials in the last 5 years. One www.futuremedicine.com

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Table 2. Recent key clinical trials targeting MUC1. Approach

PI/company

Agent

Additional Cancer agent/ adjuvant

Patients Phase (n)

Outcome

Ref.

Monoclonal antibody

Antisoma

huHMFG1 (AS1402/Therex®) Y90 mHMFG1 (666MBq/m2)

Letrazole

Breast

110

II

[302]

BSC

Ovarian

447

III

Finn OJ

100-mer (5× VNTR) unglycosylated

SB-AS2

Pancreatic

16

I

Hinoda Y

100-mer (5× VNTR) unglycosylated MUC1 (106-mer, 5 × VNTR, unglycosylated)KLH conjugate 105-mer (5 × VNTR) unglycosylated BLP25/ Stimuvax®

IFA

Pancreatic/ bile duct Breast

9

I

9

I

Low efficacy; terminated Mild side effects, no deviation from control arm; terminated Some humoral and cellular responses Some humoral responses Good humoral responses

63

I

BSC

Adenocarcinomas NSCLC

171

IIB

Apostolopoulos V Oxidised mannan-MUC1

BSC

Breast

31

Pilot III

Livingstone PO

Hexavalent vaccine (GM2, Globo-H, Lewis Y + unglycosylated, T- or Tn-MUC1)-KLH conjugate

QS-21

Prostate

30

II

Livingstone PO

Heptavalent vaccine (GM2, Globo-H, Lewis Y, Tn + STn-, T- or Tn-MUC1)KLH conjugate Autologous DCs pulsed with HER-2/MUC1 peptides Autologous DCs pulsed with MUC1/autologous tumor lysate Autologous DCs pulsed with necrotic tumor cells (+TNF-D) Autologous DC-tumor cell fusions Autologous DCs pulsed with MUC1 peptide (+TNF-D) + nonadherent cells cultured with YPK cell line (+IL-2) Autologous CD4 lymphocytes pulsed with MUC1 peptide + IL-2

QS-21

Ovarian, fallopian tube and peritoneal Breast and ovarian Breast and lung

11

I

10

I

14

I

BSC

NSCLC

8

I

BSC

23

I

BSC

Breast and renal Pancreatic

20

I

BSC

Ovarian

7

I

Antisoma

Peptidebased

Livingstone PO

Lotze MT Oncothyreon

Ex vivo leukocyte

Brossart P Fujino S

Liu KJ

Kufe D Oka M

Wright SE

QS-21

BCG

BSC BSC

Some cellular responses Improved survival; now in Phase III trials No recurrences observed in treated group; awaiting Phase III trial Frequent but low humoral responses; redesigned – see immediately below Good humoral and cellular responses Good cellular responses

[69]

[104]

[105] [106,108]

[109] [110]

[114]

[126]

[127]

[132] [134]

Frequent, low cellular responses Some cellular responses

[135]

[140] [145]

Some cellular responses

[146]

BCG: Bacillus Calmette–Guérin; BSC: Best standard of care; DC: Dendritic cell; KLH: Keyhole limpet hemocyanin; IFA: Incomplete Freund’s adjuvant; MVA: Modified vaccinia Ankara; NSCLC: Non-small-cell lung carcinoma; PSA: Prostate-specific antigen; STn: Sialyl Tn; VNTR: Variable number of tandem repeats.

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Table 2. Recent key clinical trials targeting MUC1. Approach

PI/company

Agent

Additional Cancer agent/ adjuvant

Patients Phase (n)

Outcome

Ref.

Viral vector

Transgene

MVA-MUC1-IL-2 (TG4010)

Cisplatin and NSCLC vinorelbine

44

II

Transgene

MVA-MUC1-IL-2 (TG4010)

BSC

Prostate

40

II

Therion

PANVAC-VF®

GM-CSF

Pancreatic

255

III

[155,156] Results demonstrate combination with chemotherapy is feasible; in Phase II/III trial [157] Improvement in PSA doubling time; in Phase II/III trial [304] No deviation from control arm; terminated

BCG: Bacillus Calmette–Guérin; BSC: Best standard of care; DC: Dendritic cell; KLH: Keyhole limpet hemocyanin; IFA: Incomplete Freund’s adjuvant; MVA: Modified vaccinia Ankara; NSCLC: Non-small-cell lung carcinoma; PSA: Prostate-specific antigen; STn: Sialyl Tn; VNTR: Variable number of tandem repeats.

company, Antisoma, has been using humanized HMFG1 [3] in various formulations. AS1402 (humanized HMFG1) was in a Phase II breast cancer trial in combination with letrozole, but was discontinued in August 2009 as it was unlikely to give positive efficacy findings [302] . PAM4 made it to a Phase I trial in 2007, but this trial was terminated in 2008 owing to loss of funding. A Phase III trial with Y90 -labeled murine HMFG1 was also ultimately disappointing. A total of 447 stage IC–IV ovarian cancer patients were recruited and treated with a single high dose. After a median follow-up of 3.5 years, 70 out of 224 of the treated arm and 61 out of 223 in the control arm had died, with a similar ratio of relapse. The authors strongly argue that lack of divergence was due to either the use of a single high dose rather than multiple treatments or leakage of the antibody from the peritoneum [78,79] . Overall, the anti-MUC1 monoclonal antibody clinical trials have been largely disappointing [301] . There may be several reasons for this:  Some of the most successful clinical monoclonal antibodies are multifunctional, targeting cells for killing while antagonizing the function. For example, trastuzumab (Herceptin®) inhibits ErbB2 signaling, thereby inhibiting growth, as well as opsonizing the target cell [80] . Although some of the antiMUC1 monoclonal antibodies used to date in clinical trials have been demonstrated to stimulate antibody-dependent cell-mediated cytotoxicity [81] , it is unclear whether these future science group

antibodies have an effect on MUC1 function and if targeting this function will affect tumor growth;  MUC1 levels in the sera of late-stage cancer patients can be extremely high [82,83] . If administered systemically, anti-MUC1 antibodies that target epitopes on the cleaved form may be mopped up by the serum MUC1 [81,84] . This is a major consideration when developing targeting monoclonal antibodies; however, it must be noted that, in imaging studies, anti-VNTR MUC1 antibodies were observed to reach their targets efficiently [85,86] ;  Some anticancer-associated MUC1 antibodies bind with a greater affinity or avidity compared with others, while others are not highly specific for the tumor-associated form. Those chosen for clinical trial are now not necessarily the most appropriate. As each antibody’s binding specificity is understood and agreed upon, it may be possible to target different glycoforms of MUC1, dependent on the expression by individual tumors;  Tumors are not homogenous for levels of expression and glycosylation of MUC1 [87] . Targeting of more than one TAA may be more efficacious;  The use of antibody fragments may overcome the poor penetration of full-sized monoclonal antibodies into solid tumors. Recently, intermediate- sized antibodies have been developed in large-scale recombinant protein systems that have the added benefit of low running costs. Anti-MUC1 antibody www.futuremedicine.com

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fragments (single-chain variable fragments, bivalent single-chain variable fragments and Fabs, bi- and tri-body molecules) have been produced in Pichia pastoris [88] , and transgenic tobacco plants have been engineered to produce a single-domain anti-MUC1 antibody (V[HH]) [89] .  ‘Passive’ T-cell strategies The isolation, ex vivo expansion and reinfusion of tumor-infiltrating lymphocyes has been particularly successful in the treatment of some tumors, especially melanoma [90] . However, the isolation of MUC1 tumor-infiltrating lymphocytes has met with limited success [91,92] . Crossing a murine model of spontaneous pancreatic adenocarcinoma with the MUC1 transgenic mouse (MUC1-Tg) induced naturally occurring circulating CTLs to cancer-associated MUC1 [93,94] . These MHC class I restricted CTLs were able to kill MUC1-expressing tumor cells when adoptively transferred, but the tumors were eventually able to progress by clonal expansion of cells that did not express MUC1. Furthermore, it appeared that the isolated MUC1-specific CTLs from the host animal were often anergic, probably owing to factors in the tumor microenvironment. Although it was possible to ‘reactivate’ these cells using anti-CD40 costimulation to kill target cells in vitro, effects on tumor burden were not observed [95] . Very similar results were observed in a spontaneous model of breast cancer (polyoma middle T antigen) [96] . One of the main considerations in any technique involving cytotoxic T cells is the downregulation of MHC class I observed in many cancers (between 9 and 52% depending on the tumor [97]), leading to poor CTL recognition and killing. By genetically modifying the T-cell receptor (TCR) to target surface molecules by the use of antibody fragments themselves, and the inclusion of two intracellular signaling motifs (CD3-] and CD28), it has been possible to effectively circumvent some of these issues. The antibody fragment gives specificity, and the motifs provide T-cell signals I and II, leading to clonal expansion in the absence of MHC [98] . This chimeric antigen-receptor technology has been used for targeting MUC1. Using a variety of antibody fragments fused to a TCR, transfected T cells were observed to proliferate vigorously upon repeated encounters with soluble or membraneassociated MUC1 and produce IFN-J, IL-2 and IL-17. The T cells killed MUC1+ lines in vitro and induced a significant delay in tumor growth in a xenograph mouse model [99] . 312


Active immunotherapy  Vaccination strategies The most effective and least expensive immunotherapy is always likely to be vaccination and other TAAs have successfully been targeted in this way. However, in the case of human papillomavirus and cervical cancer, for example, it took 26 years for the observation of an association to lead to a vaccine, despite having foreign antigens [100] . The use of MUC1 as a target was always going to be more difficult for a number of reasons, but not least because it is a self-antigen and tolerance must be overcome. However, if it is possible for MUC1+ patients to evoke naturally occurring MUC1-specific CTLs and antibodies, then it should be possible to promote the production of these responses via vaccination. Although humoral responses can be involved in tumor rejection and are associated with a better prognosis, it is generally accepted that the induction of CTLs, and thus presentation via MHC class I, is necessary for the generation of an efficient antitumor response. Advances in this area were initially held back by the lack of an appropriate animal model – murine Muc1 is dissimilar enough to human MUC1 for a humoral and cellular response to occur when wild-type mice are challenged with human MUC1 [101] . MUC1-Tg mouse models have since been developed that express MUC1 in a similar pattern to that in humans in noncancerous and cancerous models, and these have been used for preclinical testing of potential vaccines [102,103] . Peptide-based MUC1 vaccines

There have been a number of Phase I trials using peptides based on the VNTR of MUC1 conjugated or mixed with a variety of innate stimuli of the innate immune system. In the case of these trials, unglycosylated peptides based on various numbers of tandem repeats have been administered with various adjuvants including SB-AS2 [104] , incomplete Freund’s [105] or coupled to keyhole limpet hemocyanin (KLH) [106–108] . Humoral and cellular immune responses were observed in some cases [107,109] , but the evidence for the production of effective CTLs is less robust [104–106,108] . However, two unglycosylated VNTR formulations have been successful enough to advance to Phase III clinical trials. First, Stimuvax ® (Merck, NJ, USA) is a 25-mer VNTR MUC1 peptide in a liposome formulation with a Toll-like receptor-4 agonist (Lipid A), which is now in three Phase III future science group

MUC1 immunotherapy

trials: two in non-small-cell lung carcinoma (NSCLC) and one in estrogen receptor-positive breast carcinomas in combination with hormonal therapy. These trials are based on the encouraging results of a Phase IIB study that demonstrated that the median survival for that subset of patients with stage IIIB NSCLC (locoregional disease) was 30.6 months for patients treated with Stimuvax plus the best standard of care versus 13.3 months for the patients treated with the best standard of care alone [110] . Second, MUC1 (100-mer) coupled to oxidized mannan has been in use for many years, demon strating efficacy in preclinical studies [111,112] , Phase I and Phase II trials [113] , most recently being tested in a pilot Phase III trial. A total of 31 stage II postmenopausal breast cancer patients were immunized with mannan-MUC1 nine times over 36 weeks in combination with tamoxifen. Out of 15 patients in the placebo group there were four relapses and out of 16 patients in the oxidized mannan– MUC1-treated group there were no relapses (p = 0.0292) [114] . Although the number of patients is small, 8 years after the first treatment, no treated patient had relapsed [115] , and a full Phase III trial is hoped for soon. The key in this case may be the use of oxidized mannan, which aids internalization via the mannose receptor and so may provoke cross-presentation [112] , as well as maturing dendritic cells (DCs) [116] . In patients immunized with formulations based on the unglycosylated MUC1 TR, the antibodies detected in the patients’ sera did not consistently react with breast cancers or breast cancer cell lines [106,117] . It has been previously demonstrated that MUC1-Tn and MUC1-sialyl Tn (STn) glycopeptides can engender an effective humoral antitumor response in the MUC1-Tg mouse [118] , and that cytotoxic T cells from colorectal patients were specifically carbohydrate-dependent for MUC1-T [119] . A recent paper demonstrated that a MUC1-Tn glycopeptide was able to induce MUC1-Tn glycopeptide and glycoprotein-specific T-cell and antibody responses in both wild-type and MUC1-Tgs. The authors concluded that MUC1 glycopeptides induce stronger immunity in MUC1-Tg mice owing to the fact that they are recognized as ‘foreign’ rather than ‘self ’, and because they are preferentially cross-presented by DCs [120] . The consensus is that immunizing with glycopeptides would be ideal; however, there are issues with this approach that are discussed later in this review. future science group

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Glycans as vaccines

The changes in O-linked glycosylation that occur in carcinoma can have an effect on the phenotype and biological behavior of the tumor cells. For example, the increased expression of the STn epitope in a proportion of breast cancers (25–30%) has been related to a poor response to chemotherapy [121] . Although not the sole carrier, MUC1 is one of the major glycoproteins that can express STn. Clinical studies using STn linked to KLH (Theratope®, Biomura, Italy) were initiated some years ago [122] , culminating in an ultimately unsuccessful Phase III trial. Although the mouse data and low toxicity in Phase I and II were encouraging [122,123] , a randomized, doubleblind, Phase III trial of 1030 women across 120 international centers failed to demonstrate a significant effect versus the control vaccine [124] . However, it must be noted that STn expression was not evaluated as a prerequisite for study entry, and therefore the possibility of a benefit in a subset of patients whose tumors expressed STn could not be excluded. Recently, when a suitable mouse model was used, Theratope was found to be successful in promoting antibody-mediated inhibition of tumor growth by targeting multiple STn-carrying proteins, not just MUC1 [125] . Building on this concept, a new hexavalent construct was developed and entered into a Phase II trial. The vaccine included GM2, Globo H, Lewis Y, glycosylated MUC1–32-mer and Tn and TF (T) in a clustered formation, conjugated to KLH and mixed with QS-21. A total of 30 patients with advanced prostate cancer were treated with eight vaccinations over 13 months. This hexavalent vaccine of synthetic ‘self ’ antigens broke immunologic tolerance against two or more antigens in all 30 vaccinated patients. It was safe, but antibody titers against several of the antigens were lower than those seen in individual monovalent trials. The authors suggest that this may be due to the immunosuppressive effects of MUC1 used in their formulation [126] . This work was followed by a pilot trial using a heptavalent construct, which includes the glycopeptides MUC1-Tn, T and STn with the unglycosylated form removed, on 11 patients with adenocarcinomas. Humoral responses were more potent and some cellular responses were observed, with a further trial now being hoped for [127] . Dendritic cells

Dendritic cell vaccination strategies have been widely pursued over the last 10 years, owing to a recognition of the importance of these cells www.futuremedicine.com

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in linking the innate and adaptive immune responses. DCs are the most potent antigenpresenting cells of the body and are able to present exogenous antigens on either MHC class II, or cross-present exogenous antigens on MHC class I to T cells [128] . One study compared MUC1 VNTR vaccination strategies in a mouse model, finding that only the pulsed DCs were able to provoke a specific CTL response [129] . There are three major ways in which DCs have been used in MUC1 immunotherapy. They have been pulsed ex vivo with peptides [130–132] or cell lysates [133–135] , fused with tumor cells [136–141] or transfected with RNA [142,143] . DCs pulsed ex vivo with MUC1 peptides have been used in a number of early clinical trials, with some degree of success [130–132] . Recent work has focused on improving on these findings. By pulsing DCs with MUC1 peptide and administering these cells with the pan-HLA-DR binding peptide, PADRE, increased HLA-A2 mediated MUC1specific CTL killing (with increased IL-12 and IFN-J) was observed in vitro [144] . A recent study pulsed DCs with MUC1 peptide and TNF-D while simultaneously culturing the nonadherent cell population with a pancreatic cell line (YPK1) and IL-2. Both the MUC1-pulsed DCs and MUC1 lymphocytes were injected into pancreatic carcinoma patients between two and 15-times. One patient with multiple lung metastases demonstrated complete response and another five patients demonstrated stable disease [145] . In a similar vein, one group has successfully increased ovarian cancer patients’ mean survival simply by reinfusing autologous T cells after stimulation with MUC1 peptide and IL-2 [146] . The fusion of mature or immature DCs with autologous/nonautologous tumor cells, effectively causing the DCs to present a wide variety of tumor antigens as well as costimulatory molecules and cytokines, has also been used in early clinical studies. Using this method, anti-MUC1 CD4 and CD8 T cells have been generated and disease regression observed [140,141] . However, it has recently been demonstrated that without the appropriate adjuvants, efficacy using this method can be grossly affected by regulatory T-cell induction [147] . Readers are referred to a review in a 2009 edition of Immunotherapy for a wider view of this area [148] . Another method of inducing DCs to express a particular TAA is by transfecting them with the RNA of the antigen in question. In use since the mid-1990s, this technique has been observed to elicit tumor-specific CTLs in many scenarios. In experiments relevant to this review, 314


DCs transfected with MUC1 RNA were seen to evoke class I- and class II-restricted T-cell responses against MCF7 cells and a MUC1+ renal carcinoma cell line [149] . In an in vitro lung cancer model, immature DCs transfected with total lung carcinoma RNA were observed to express both MUC1 and CEA and initiate a CTL tumor-specific response against relevant cell lines [150] . The majority of this DC work is encouraging; however, it must be noted that, in mice, it has been observed that naive unpulsed autologous DCs alone can elicit an antitumor response in a tumor challenge model [151] . Thus, it should be remembered that, until the stage is reached when these type of trials have a large control arm, simply manipulating DCs ex vivo may lead to nonspecific activation. MUC1 cDNA

The use of cDNA as a form of vaccination has only recently begun in clinical trials and has not progressed beyond Phase I/II, mainly owing to the absence of a potent immunological response. The current cDNA trials in non-MUC1 studies appear to be focusing on different cDNA delivery systems and dose-escalation trials [301] – it would appear that, until there are profound responses seen in the human model, the scientific consensus is that there are more controllable and effective methods of presentation. If these issues are resolved, the MUC1 cDNA preclinical studies are encouraging – intramuscular injection of MUC1 cDNA has successfully protected mice in several studies [152,153] . Viral vectors

The majority of mouse and patient work has used vaccinia; however, a replicationincompetent adenovirus vector has recently entered trials, using the whole human MUC1 cDNA plus a CD40L adjuvant [301] . Vaccinia can efficiently infect a variety of cell types and its use can cause localized inflammation at the injection site, as well as influenze-like symptoms, and as such, an attenuated form (modified vaccinia Ankara [MVA]), which is unable to replicate, is deemed safer. Following successful Phase I and II trials of MVA-expressing MUC1 and IL-2 (TG4010) in NSCLC and breast cancer [154,155] , the company Transgene (France) have taken this formulation into a Phase II/III trial. In the Phase II study, TG4010 was administered simultaneously with cisplatin and vinorelbine in 44 stage IIIB/IV patients with NSCLC. future science group

MUC1 immunotherapy

This chemo–immunotherapy regime achieved a tumor response rate of 37%. Importantly, patients with a MUC1-specific cellular immune response had a significantly better overall survival (p = 0.0035) [156,303] . TG4010 has also been used in a Phase II trial of prostate cancer. In this study, the prostate-specific antigen levels were measured in the patients, with 63% having a significant increase in prostate-specific antigen doubling time [157] . A MVA virus expressing CEA, MUC1 and TRICOM (ICAM-1, B7.1 and LFA-3), known as PANVAC™-VF, has also been used in Phase I/II trials of pancreatic, colorectal and breast cancer patients, leading to a Phase III trial in advanced pancreatic carcinomas. A total of 255 patients were recruited by 2006 and treated with PANVAC-VF (Therion, MA, USA) plus GM-CSF; however, a press release in 2009 indicated that there were no deviations from the control arm and hence the trial was deemed unsuccessful, ultimately triggering the sale of Therion [304] . Previous publications had demonstrated that some T-cell responses were observed, with an associated increase in survival in those responders [158,159] .

Generation of MUC1-specific T cells by vaccination: the problems Vaccination strategies require exogenous antigens to be securely displayed on MHC class I or II molecules on APCs in order to bind specific TCRs. The mechanism of class II loading is well established, as is the presentation of endogenous antigen on class I. However, binding exogenous antigen on MHC class I, and stimulation of CTLs, requires cross-presentation. The mechanisms of loading exogenous antigen onto class I and II MHC are summarized in FIGURE 4. The letters below refer to this figure. (A) The epitopes in the VNTRs make up a large domain of the MUC1 structure, with each repeat containing 20 amino acids. CTLs from breast cancer patients have been observed to react to a range of MUC1 epitopes, not just in the VNTR [50,51,160] , thus several groups are now adopting strategies that encourage presentation of all possible epitopes [157,158] . (B) Internalization of antigen and MHC pathway determination. In APCs, exogenous cancerassociated MUC1 can be internalized for potential presentation on MHC class II via the classical pathway. As previously mentioned, for a CTL response, MUC1 peptides must be presented on MHC class I by cross-presentation. The efficacy of this process depends upon the type of APC [161,162], future science group

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the lectin that is bound [163] and the type of glycosylation on MUC1 [66,67] . Cancer-associated MUC1 may actually be more readily taken up by APCs, as it expresses clustered polyanions (sialic acid groups) and truncated carbohydrates, some of which are ligands for endocytotic C-type lectins and scavenger receptors. (C) Inhibition of proteolysis. Once inside the immunoproteasome, MUC1 needs to be cleaved into small peptides. However, O-linked glycans are known to protect peptide backbones from such degradation since it is key to their function. As such, the cleavage (and subsequent presentation) of noncancerous MUC1, with its long carbohydrate chains, is unlikely to occur. However, cleavage of the truncated, cancerous glycoproteins has been found to be possible, with certain provisos [164] ; recently, immunoproteasomes have been found to be able to cleave MUC1 VNTR glycopeptides if they carry GalNAc and Gal– GalNAc residues [165] . However, the glycans can only be tolerated in specific locations, and cleavage in the presence of sialic acid has never been observed. In the class II context, it appears that O-glycosylated proteins can be processed via the classical pathway in vitro [166] , with cleavage again being dependent on the glycan location and structure. In conclusion, it appears that the cleavage of MUC1-Tn and, to some extent, MUC1-T is possible, but is heavily dependent on the VNTR site of glycosylation. (D) Binding of glycopeptides to the MHC. Once a restricted array of glycopeptides is produced by cleavage, can these bind to the MHC? Crucially, cytosolic and nuclear glycopeptides have been observed on MHC class I molecules [167] , so, logically, it may be possible to load mucinous O-linked glycoproteins or glycopeptides for presentation while preserving their glycans. MHC class I molecules have been found to bind unglycosylated MUC1 [160] and MUC1-Tn glycopeptides [168] , in one instance with a greater affinity than the unglycosylated form [169] . However, the addition of a E1,3-linked galactose prevented this binding [165] . MUC1-Tn glycopeptides have also been found to bind MHC class II molecules, with the glycans being demonstrated to point away from the groove, suggesting a possible mechanism for glycan-specific T-cell responses [170] . It must be noted that any of these interactions need to be strong, as the peptide must remain in place to be ready for recognition by a specific TCR in the lymphatics [171] . (E) Interaction with the TCR. Once in the lymph node, effective interaction between the MHC-loaded peptide and the TCR must occur www.futuremedicine.com

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MUC1

Digested MUC1 Exogenous antigen

Classical MHC class I pathway

Phagosome/endosome

Key

Nonclassical MHC class I pathway

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Phagolysosome

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Immunotherapy © Future Science Group (2010)

Figure 4. Antigen processing and MHC loading in an antigen-presenting cell. Letters refer to descriptions in the text. Class II presentation: the classical presentation of exogenous antigen, requiring antigen-presenting cells to internalize the antigen via receptormediated endocyotosis or pinocytosis, before proteolysis in specialized endosomal compartments, resulting in the production of antigenic epitopes for display on MHC class II molecules to CD4 T cells. Class I presentation: the classical presentation of endogenous antigen requiring cleavage of the antigen in the immunoproteasome, resulting in the production of peptides that are transported by TAP to the ER in order to bind to MHC class I molecules. These molecules are then expressed on the cell surface where they can engage with the T-cell receptor expressed by CD8 T cells. Cross-presentation: exogenous antigen enters the class I endogenous pathway either by direct entry into the cytosol or escape from endosomal compartments. Once in the cytosol, it can enter the immunoproteasome and be presented as if endogenous. ER: Endoplasmic reticulum; TAP: Transporter of antigenic peptide; TCR: T-cell receptor.

for effective T-cell stimulation. One group found that any glycosylation at the VTSA or PDTR regions blocked the activation of a hybridoma with a TCR-specific for unglycosylated MUC1 – the glycans appeared to block interaction – feeding into the theory of TCR glycopeptide specificity [172,173] . Ultimately, MUC1-derived glycopeptides (or glycopeptides expressing core-1 glycans on an alternative backbone [174] ) have been found to elicit glycopeptide-specific CD8 + T-cell responses in cultured splenocytes or following immunization of experimental animals [120,168,175] , demonstrating that with the 316


right set of conditions, it may be possible to elicit carbohydrate-specific cancer-associated MUC1 responses in patients [176] .

Epitope presentation by the target cell For an effective recognition by CTLs, the target cell needs to load epitopes that correspond to those displayed by APCs onto MHC class I in order to mark itself out for killing. One group found that, although it was possible to induce MUC1-Tn CTL responses in MUC1-Tgs, they were less effective than hoped, partially owing to the low levels of MUC1-Tn expressed, and future science group

MUC1 immunotherapy

therefore presented, on the target cells [168] . Presentation of endogenous cancer-associated MUC1 is currently an under-investigated area; however, drawing from other fields, there are likely to be three main mechanisms:  Through the normal endogenous antigen pathway via the ubiquitination of recycled material and progression through the MHC class I machinery;  Through the ER-associated protein degradation–cytosol pathway, where misfolded proteins leave the ER into the cytosol before being presented on MHC class I. This has been observed in renal cell carcinoma (FGF-5 [177]) and in melanoma (gp100 [178]). Importantly, once proteins reach the Golgi, where the O-glycans are added, this route is closed, meaning that only unglycosylated peptides could be presented in this manner;  Through a novel internalization mechanism. A recent publication using melanoma as a model demonstrates an exogenous antigen (matrix metalloproteinase 2) secreted by the tumor cell itself being internalized before passing into the proteasome via the cytosol [179] . Furthermore, endogenous antigen has now been observed to be expressed on MHC class II via autophagy [180] – the systems appear to be far more fluid than thought. The MMP2 paper opens up the possibility that secreted or membrane-bound MUC1 could be internalized and presented on MHC class I, and, critically, may retain its surface glycosylation. The organelles responsible for protein cleavage and epitope production in the class I context are proteasomes and immunoproteasomes. Under normal conditions, proteasomes are found in most MHC class I-expressing cells, whereas cells of the immune system contain immunoproteosomes. Each component in each cell type houses a different set of enzymes, which may result in a different profile of displayable class I peptides [181] . For an effective CTL response, MHC class I-presented epitope on the target cell should be the same as that displayed on the APC that induces the immune response. At present, the efficacy of tumor cells for displaying glycosylated peptides needs to be proven, and a better understanding of this area may benefit MUC1 immunotherapy enormously [182,183] . Intriguingly, the presence of non-MHCrestricted, MUC1-specific T cells in cancer patients was reported a number of years ago [184,185] , and recently another group has future science group

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published regarding the same phenomenon [186] . It is hypothesized that the MUC1 VNTR– TCR interactions and subsequent signaling may replace the MHC-loaded antigen–TCR signal [187] . These non-MHC-restricted TCRs have been isolated from a T-cell clone, sequenced, genetically modified and transfected into a variety of immune cells, demonstrating efficacy in two mouse models [188,189] . However, until this novel and exciting immunological mechanism is robustly proven and universally accepted, there will be understandable uncertainties within the wider immunological community.

Additional considerations  Autoimmunity As we have discussed, a large variety of techniques to target cancer-associated human MUC1 have been tested in mice and humans, and, to date, no cases of autoimmunity have been reported, which is reassuring.  Tumor microenvironment Any immunological method used to target MUC1 must be proven to be effective in the tumor microenvironment. With the carcinoma actively skewing the system to anergy or, failing that, a humoral response, it is a difficult environment in which to mount a potent cellular immune attack [190,191] . Indeed, the induction of MUC1specific CTLs has been observed to correlate with tumors of high differentiation, ER expression and low proliferative activity, while the degree of this differentiation of the tumors correlated with high local IFN-D and low local TGF-E1. Interestingly, the presence of MUC1-specific B-cell responses (mainly IgM) were found in patients with poorly differentiated advanced tumors, which were characterized by increased local TGF-E1, reduced local IFN-D and the absence of CTL responses. The CTL responses correlated with a good prognosis while the B-cell responses did not [192] . Interestingly, a recent Phase I/II trial induced ex vivo MUC1-specific autologous CTLs and reinfused them into four previously treated metastatic breast cancer patients. The CTLs generated from patients with high tumor burden had lower cytokine production and cytotoxicty against MCF7 cells than those from patients in remission [193] .  Immunosuppressive effects of MUC1 Sialyl Tn-expressing mucins have been known to inhibit natural killer cell cytotoxicity for a number of years [194,195] , and evidence is building for the immunomodulative effects of mucins expressing www.futuremedicine.com

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this glycan [196,197] . However, the immune effects of the other cancer-associated glycoforms have only recently been documented. Using a transplantable tumor model, we have observed that murine tumors carrying sialylated core-1-based O-glycans grow significantly faster than identical tumors carrying core-2-based O-glycans. As this effect was not observed in immunodeficient mice, the mechanism involved was thought to be immunological [198] . Further work has suggested that cancer-associated MUC1 induces a switch to the humoral or anergic responses, rather than the cellular response [199–205] . More knowledge is needed regarding the immunological effects of specific MUC1 glycoforms in order to establish if there is any modulation: this is an important consideration when designing MUC1-based immunotherapeutic agents.

Future perspective  Target function The evidence of 15 years of anticancer-associated MUC1 monoclonal antibody therapy suggests that we need an antibody which is multifunctional, such as trastuzumab, and although MUC1 may be a poorer proliferative driver than ErbB2, it has multiple intracellular mechanisms that, in combination, may be important for tumor growth and prognosis [63] . One group is investigating the post-cleavage MUC1-D/E junction as an antibody target, with some success in a xenograft model [206] . However, it should be noted that this region must be treated with caution, since it has been observed to stimulate growth in a ligand-dependent [207] and -independent manner [208] . Another target may be the intracellular CT: small peptides have been observed to block MUC1’s intracellular interactions, inhibiting the pro-proliferative effects in vitro and in vivo [209,210] .  Novel delivery mechanisms Exosomes are small membrane vesicles that are released into the extracellular environment during fusion of multivesicular bodies with the plasma membrane. They are secreted by various cell types including hematopoietic cells, normal epithelial cells and even some tumor cells. Being small samples of the membrane, they are known to carry MHC class I and various costimulatory molecules when produced by APCs, and are readily able to fuse with the membranes of other cells. Given this, their potential for immunotherapy as a potent delivery system is clear [211] . Using mouse cell lines transfected with human MUC1, one group 318


purified the resultant exosomes and were able to demonstrate that both autologous and allogenic exosome vaccinations were able to stimulate immunity and suppress the growth of human MUC1-expressing tumor cells in vivo [212] . Many believe the field has great potential once issues with the generation of specific exosomes and their delivery are resolved.  Chimeric antigen receptors Chimeric antigen receptor technology negates many of the problems associated with CTLbased therapies by targeting the unprocessed molecule. However, the main problem is expense: GMP-grade isolation, transfection and expansion costs are high. Toxicity is also a concern when targeting depends on a single selfprotein with several solutions being explored. For example, the targeting of two tumor antigens with separate signaling motifs per antigen may increase specificity.  Selecting or inducing cross-presentation in antigen-presenting cells It may be possible to target DCs in vivo and induce cross-presentation by delivering antigen via lectin-like receptors that are associated with this process (e.g., DEC205 [213] and MMR [163]) or pharmacologically inducing a high pH in the phagosome [214] . Furthermore, it should be noted that not all DCs cross-present to the same extent; a subset, commonly called CD8D DCs, which appear to be specialized for this task, has been identified in mice. Similar subsets of DCs appear to exist in humans and it may be possible to target these cells in vivo through the lectin DNGR-1 [161] . Moreover, by treating cells/patients with chloroquine, it may be possible to promote cross-presentation through the transfer of endocytosed material into the cytosol [215] . JG T-APC cells have also been found to crosspresent tumor and microbial antigens [162] , as have B cells in MUC1-Tg mice, thereby breaking tolerance [216] . If primed, these cells may be able to recruit specific CTLs in addition to their already characterized antitumor effects [217] , potentially making them ‘effector-managers’ of the immune system.  Combination therapy Combination therapies will almost certainly have their place in future strategies. Chemotherapy and radiotherapy are ideal immunological adjuvants, as both induce future science group

MUC1 immunotherapy

many additional TAAs for APCs to present. Sublethal irradiation of various tumor cell lines enhances MUC1 CTL killing in vitro by modifying their phenotype [218] , while in head and neck squamous cell carcinoma, cells treated with multiagent chemotherapy and radiotherapy were observed to be more sensitive to perforin [219] . On the negative side, both therapies effect dividing cells directly and therefore the clonal expansion of T and B subsets. In addition, there is evidence that chemo- and radio-therapy may potentiate the tumor microenvironment via other mechanisms [220,221] .

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Pharmacological approaches, such as COX-2 inhibition, as well as additional adjuvants, also appear to be promising in preclinical models [147,222–224] .

Conclusion The pathway required to carry an immunotherapeutic strategy from the bench to the clinic is not straightforward. Patients with advanced disease are commonly recruited for Phase I and II trials, and individuals with this level of disease burden often do not have a fully operational immune system. As such, it is rarely toxicity that

Executive summary MUC1 as an immunotherapeutic target MUC1 is upregulated and aberrantly glycosylated in the majority of adenocarcinomas. Natural immune responses to MUC1 Humoral and cellular responses have been observed and the presence of a response is associated with an improved prognosis. Function of MUC1 Signaling through the intracellular domain has been found to facilitate and promote tumor function. MUC1 immunotherapy: passive Monoclonal antibodies: many preclinical trials demonstrated good efficacy; however, very few effects were seen in clinical trials, including one Phase III trial. These studies focused on the tandem repeat region. Passive T-cell strategies: the expansion of tumor-infiltrating lymphocyes has proved unsuccessful; however, the use of genetically modified T cells to target MUC1+ tumors in a non-MHC-restricted manner has proven effective in preclinical models. MUC1 immunotherapy: active Clinical trials have been successful in inducing humoral responses; however, it has proved more difficult to promote cytotoxic T lymphocyte production. Nevertheless, there are at least three formulations of MUC1 that have demonstrated enough potential to be taken to latestage clinical trials. MUC1 in a liposome formulation is in Phase III trials, the use of oxidized mannan as delivery system has completed a pilot Phase III trial and vaccinia virus-expressing MUC1 has completed a successful Phase IIB trial and is currently in a Phase II/III trial. An approach to target the aberrant glycosylation independently of the protein backbone has been used in five trials. Efficacy in one Phase III trial was low, possibly owing to trial design. Autologous dendritic cells have been used in three ways in order to target MUC1: pulsed with MUC1 peptides or tumor lysates; fused with tumor cells; and transfected with RNA. All are in, or have been in, multiple clinical trials, with none progressing past Phase II to date. Generation of MUC1-specific cytotoxic T lymphocytes by vaccination: the problems The main issues are limited epitopes (most peptide strategies use the tandem repeat region thus limiting the number of displayed epitopes); inhibition of proteolysis (the most specific responses involve using glycopeptides; however, glycans can block proteolysis); and binding to MHC molecules (the glycans can affect binding in a positive or negative manner). Epitope presentation by the target cell For an effective cytotoxic T lymphocyte response, it is critical that the displayed epitopes on the antigen-presenting cell (APC) and target cell match. The presentation of endogenous MUC1 glycopeptides on the target cell is an under-researched area. Additional considerations Autoimmunity: there have been no reported cases of autoimmunity in any trial to date. The tumor microenvironment: MUC1 immunotherapy, similar to most immunotherapy, has been observed to be affected by the microenvironment. The immunosuppressive effects of MUC1: there is growing evidence that some cancer-associated glycoforms of MUC1 may be immunosuppressive. Future perspective Target function: using monoclonals to affect function as well as opsonizing the target cell, or using small peptides to block intracellular signaling. Novel delivery mechanisms: using exosomes to transfer cancer-associated MUC1 and stimulatory molecules to cells. Chimeric antigen receptors: targeting MUC1+ cells using this technique may prove to be extremely effective. Selecting or inducing cross-presentation in APCs by targeting APCs that cross-present, or by delivering peptides to specific receptors in vivo may prove to be effective, as could administration of agents such as chloroquine, which have been found to promote cross-presentation. Combination therapies: using chemo- or radio-therapies alongside MUC1 immunotherapy may prove to be very effective, as these strategies can synergize to great effect.



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undermines early immunotherapeutic trials, it is almost always the lack of a significant immune response. A detailed understanding of antigen processing and presentation of glycosylated proteins, such as MUC1, would enable the issues discussed in this review to be addressed, facilitating the rational design of new and more potent vaccination strategies, which may demonstrate greater effects in these patient groups. However, it must be noted that a preventative vaccine in the truest sense of the word (rather than the curative options discussed in this review) is undesirable owing to the potential long-term side effects (cancer-associated MUC1 is occasionally found in healthy circumstances). However, it may be worth considering the vaccination of high cancer-risk populations (e.g., those with mutated BRCA1/2) who do not wish to opt for a bilateral mastectomy. An additional issue will be to decipher and counteract mechanisms of MUC1-dependent tumor resistance, immunological escape and tumorigenic signaling. As such, it may be proven that, by targeting MUC1’s signaling, combining immunotherapy with chemo-, radio- or hormonal therapies, double-targeting TAAs to increase specificity or by using more specific/powerful adjuvants, we will arrive at the best clinical outcome. Bibliography Papers of special note have been highlighted as: of interest of considerable interest 1

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Acknowledgements Figure 1 was kindly supplied by Nadine Bizouarne from Transgene SA, Boulevard Gonthier d’Andernach, Parc d’Innovation – CS80166, F-67405 Illkirch-Graffenstaden, Strasbourg, France. Figure 2 was kindly imaged by Lucienne Cooper of the Breast Cancer Biology Group, London, UK. We would like to thank Ms Gursharn Hutchins for invaluable administrative support.

Financial & competing interests disclosure This work was supported by Cancer Research UK (registered charity No. 1089464. 61 Lincoln’s Inn Fields, London WC2A 3PX, UK) and King’s College London (The Strand, London WC2R 2LS, UK). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Overarching piece that is an excellent introduction to the subject and outlines much of its history. Mcauley JL, Linden SK, Png CW et al.: MUC1 cell surface mucin is a critical element of the mucosal barrier to infection. J. Clin. Invest. 117(8), 2313–2324 (2007).

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Greenlee RT, Hill-Harmon MB, Murray T, Thun M: Cancer statistics, 2001. CA Cancer J. Clin. 51(1), 15–36 (2001). Arklie J, Taylor-Papadimitrious J, Bodmer W, Egan M, Millis R: Differentiation antigens expressed by epithelial cells in the lactating breast are also detectable in breast cancers. Int. J. Cancer 28(1), 23–29 (1981).

Finally, it is reassuring to note that cancer stem cells may express cancer-associated MUC1 [225] , which leads us to hope that a successful MUC1 immunotherapy may also counteract recurrence and that, of the 62 MUC1 targeting trials researched during the course of this review, eight were Phase III (12%) and, of these trials, four are active and two are due to publish very soon [301] .


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Clear and concise review of the intracellular signalling pathways of MUC1. The diagrams are extremely useful for illustrating the main points. Hattrup CL, Gendler SJ: Structure and function of the cell surface (tethered) mucins. Annu. Rev. Physiol. 70, 431–457 (2008). Zotter S, Hageman PC, Lossnitzer A et al.: Monoclonal antibodies to epithelial sialomucins recognize epitopes at different cellular sites in adenolymphomas of the parotid gland. Int. J. Cancer Suppl. 3, 38–44 (1988).

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Wykes M, Macdonald KP, Tran M et al.: MUC1 epithelial mucin (CD227) is expressed by activated dendritic cells. J. Leukoc. Biol. 72(4), 692–701 (2002).

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38

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39

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PERSPECTIVE

Mukherjee P, Tinder TL, Basu GD, Pathangey LB, Chen L, Gendler SJ: Therapeutic efficacy of MUC1-specific cytotoxic T lymphocytes and CD137 co-stimulation in a spontaneous breast cancer model. Breast Dis. 20, 53–63 (2004).

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Wilkie S, Picco G, Foster J et al.: Retargeting of human T cells to tumorassociated MUC1: the evolution of a chimeric antigen receptor. J. Immunol. 180(7), 4901–4909 (2008).

110 Butts C, Murray N, Maksymiuk A et al.:

96

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100 Arbyn M, Dillner J: Review of current

knowledge on HPV vaccination: an appendix to the European guidelines for quality assurance in cervical cancer screening. J. Clin. Virol. 38(3), 189–197 (2007).

101 Spicer AP, Parry G, Patton S, Gendler SJ:

Molecular cloning and analysis of the mouse homologue of the tumor-associated mucin, MUC1, reveals conservation of potential O-glycosylation sites, transmembrane, and cytoplasmic domains and a loss of minisatellite-like polymorphism. J. Biol. Chem. 266(23), 15099–15109 (1991). 102 Peat N, Gendler SJ, Lalani N, Duhig T,

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111 Lees CJ, Apostolopoulos V, Acres B et al.:

Immunotherapy with mannan-MUC1 and IL-12 in MUC1 transgenic mice. Vaccine 19(2–3), 158–162 (2000). 112 Apostolopoulos V, Barnes N, Pietersz GA,

Mckenzie IF: Ex vivo targeting of the macrophage mannose receptor generates anti-tumor CTL responses. Vaccine 18(27), 3174–3184 (2000).

103 Graham RA, Morris JR, Cohen EP,

Taylor-Papadimitriou J: Up-regulation of MUC1 in mammary tumors generated in a double-transgenic mouse expressing human MUC1 cDNA, under the control of 1.4-kb 5´ MUC1 promoter sequence and the middle T oncogene, expressed from the MMTV promoter. Int. J. Cancer 92(3), 382–387 (2001). 104 Ramanathan RK, Lee KM, Mckolanis J et al.:

Phase I study of a MUC1 vaccine composed of different doses of MUC1 peptide with SB-AS2 adjuvant in resected and locally advanced pancreatic cancer. Cancer Immunol. Immunother. 54(3), 254–264 (2005). 105 Yamamoto K, Ueno T, Kawaoka T et al.:

MUC1 peptide vaccination in patients with advanced pancreas or biliary tract cancer. AntiCancer Res. 25(5), 3575–3579 (2005). 106 Gilewski T, Adluri S, Ragupathi G et al.:

Vaccination of high-risk breast cancer patients with mucin-1 (MUC1) keyhole limpet hemocyanin conjugate plus QS-21. Clin. Cancer Res. 6(5), 1693–1701 (2000).


Results from a Phase IIB trial using Stimuvax® on 171 patients with non-smallcell lung carcinoma, providing the impetus for three Phase III trials. Demonstrates that the treated group survived an average of 4.4 months longer than the control arm and that one subset of patients (those with stage IIIB locoregional disease) benefited greatly, surviving, on average, 2 years longer.

113 Karanikas V, Lodding J, Maino VC,

Mckenzie IF: Flow cytometric measurement of intracellular cytokines detects immune responses in MUC1 immunotherapy. Clin. Cancer Res. 6(3), 829–837 (2000). 114 Apostolopoulos V, Pietersz GA, Tsibanis A

et al.: Pilot Phase III immunotherapy study in early-stage breast cancer patients using oxidized mannan-MUC1 [ISRC TN71711835]. Breast Cancer Res. 8(3), R27 (2006).

Pilot Phase III study of 31 stage II breast cancer patients producing immunological and clinical effects: none out of 16 relapses in the treated group compared with four out of 15 in the control group and anti-variable number of tandem repeat (VNTR) antibodies were observed in nine out of 13 treated patients with four out of ten producing MUC1-specific cytotoxic T lymphocytes.

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115 Tang CK, Katsara M, Apostolopoulos V:

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Mannan derivatives induce phenotypic and functional maturation of mouse dendritic cells. Immunology 118(3), 372–383 (2006). 117 Kotera Y, Fontenot JD, Pecher G,

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Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptides elicit cancer-specific anti-MUC1 antibody responses and override tolerance. Glycobiology 16(2), 96–107 (2006). 119 Bohm CM, Mulder MC, Zennadi R et al.:

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Finn OJ: Tumor-associated MUC1 glycopeptide epitopes are not subject to self-tolerance and improve responses to MUC1 peptide epitopes in MUC1 transgenic mice. Biol. Chem. 390(7), 611–618 (2009). 121 Miles DW, Happerfield LC, Smith P et al.:

Expression of sialyl-Tn predicts the effect of adjuvant chemotherapy in node-positive breast cancer. Br. J. Cancer 70(6), 1272–1275 (1994). 122 Miles D, Papazisis K: Rationale for the

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with theratope (STn-KLH) as treatment for breast cancer. Expert Rev. Vaccines 3(6), 655–663 (2004).

127 Sabbatini PJ, Ragupathi G, Hood C et al.:

Pilot study of a heptavalent vaccine-keyhole limpet hemocyanin conjugate plus QS21 in patients with epithelial ovarian, fallopian tube, or peritoneal cancer. Clin. Cancer Res. 13(14), 4170–4177 (2007). 128 Nencioni A, Grunebach F, Schmidt SM

et al.: The use of dendritic cells in cancer immunotherapy. Crit. Rev. Oncol. Hematol. 65(3), 191–199 (2008). 129 Soares MM, Mehta V, Finn OJ: Three

different vaccines based on the 140-amino acid MUC1 peptide with seven tandemly repeated tumor-specific epitopes elicit distinct immune effector mechanisms in wild-type versus MUC1-transgenic mice with different potential for tumor rejection. J. Immunol. 166(11), 6555–6563 (2001). 130 Loveland BE, Zhao A, White S et al.:

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Taylor-Papadimitriou J, Noll T: Breast carcinoma cell lysate-pulsed dendritic cells cross-prime MUC1-specific CD8 + T cells identified by peptide-MHC-class-I tetramers. Cell. Immunol. 231(1–2), 112–125 (2004). 134 Kontani K, Taguchi O, Ozaki Y et al.:

Dendritic cell vaccine immunotherapy of cancer targeting MUC1 mucin. Int. J. Mol. Med. 12(4), 493–502 (2003). 135 Chang GC, Lan HC, Juang SH et al.:

A pilot clinical trial of vaccination with dendritic cells pulsed with autologous tumor cells derived from malignant pleural effusion in patients with late-stage lung carcinoma. Cancer 103(4), 763–771 (2005). 136 Chen D, Xia J, Tanaka Y et al.:

vaccine induces antibody-mediated tumour protection in a relevant murine model. Br. J. Cancer 100(11), 1746–1754 (2009).

Immunotherapy of spontaneous mammary carcinoma with fusions of dendritic cells and mucin 1-positive carcinoma cells. Immunology 109(2), 300–307 (2003).

126 Slovin SF, Ragupathi G, Fernandez C et al.:

137 Koido S, Tanaka Y, Chen D, Kufe D, Gong J:

125 Julien S, Picco G, Sewell R et al.: Sialyl-Tn

A polyvalent vaccine for high-risk prostate patients: ‘are more antigens better?’. Cancer Immunol. Immunother. 56(12), 1921–1930 (2007)

324

The kinetics of in vivo priming of CD4 and CD8 T cells by dendritic/tumor fusion cells in MUC1-transgenic mice. J. Immunol. 168(5), 2111–2117 (2002).


138 Tanaka Y, Koido S, Chen D, Gendler SJ,

Kufe D, Gong J: Vaccination with allogeneic dendritic cells fused to carcinoma cells induces antitumor immunity in MUC1 transgenic mice. Clin. Immunol. 101(2), 192–200 (2001). 139 Gong J, Apostolopoulos V, Chen D et al.:

Selection and characterization of MUC1specific CD8 + T cells from MUC1 transgenic mice immunized with dendritic-carcinoma fusion cells. Immunology 101(3), 316–324 (2000). 140 Avigan D, Vasir B, Gong J et al.: Fusion cell

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Processing and presentation of HLA class I and II epitopes by dendritic cells after transfection with in vitro-transcribed MUC1 RNA. Blood 105(8), 3199–3205 (2005). 144 Brossart P, Heinrich KS, Stuhler G et al.:

Identification of HLA-A2-restricted T-cell epitopes derived from the MUC1 tumor antigen for broadly applicable vaccine therapies. Blood 93(12), 4309–4317 (1999). 145 Kondo H, Hazama S, Kawaoka T et al.:

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149 Muller MR, Grunebach F, Nencioni A,

Brossart P: Transfection of dendritic cells with RNA induces CD4- and CD8-mediated T cell immunity against breast carcinomas and reveals the immunodominance of presented T cell epitopes. J. Immunol. 170(12), 5892–5896 (2003).

158 Kaufman HL, Kim-Schulze S, Manson K

152 Plunkett T, Graham R, Correa I et al.:

Protection against MUC1 expressing mouse tumours by intra-muscular injection of MUC1 cDNA requires functional CD8 + and CD4 + T cells but does not require the MUC1 tandem repeat domain. Int. J. Cancer 109(5), 691–697 (2004).

study of vaccination with recombinant CEA-MUC-1-TRICOM poxviral-based vaccines in patients with metastatic carcinoma. Clin. Cancer Res. 14(10), 3060–3069 (2008).

170 Glithero A, Tormo J, Haurum JS et al.: Crystal

160 Domenech N, Henderson RA, Finn OJ:

Functional avidity of tumor antigen-specific CTL recognition directly correlates with the stability of MHC/peptide multimer binding to TCR. J. Immunol. 168(3), 1167–1171 (2002).

Identification of an HLA-A11-restricted epitope from the tandem repeat domain of the epithelial tumor antigen mucin. J. Immunol. 155(10), 4766–4774 (1995).

154 Scholl S, Squiban P, Bizouarne N et al.:

Metastatic breast tumour regression following treatment by a gene-modified vaccinia virus expressing MUC1 and IL-2. J. Biomed. Biotechnol. 2003(3), 194–201 (2003). 155 Acres B: Cancer immunotherapy: Phase II

clinical studies with TG4010 (MVA-MUC1IL2). J. BUON 12(Suppl. 1), S71–S75 (2007). 156 Ramlau R, Quoix E, Rolski J et al.: A Phase II

study of Tg4010 (MVA-MUC1-IL2) in association with chemotherapy in patients with stage III/IV non-small cell lung cancer. J. Thorac. Oncol. 3(7), 735–744 (2008). 157 Dreicer R, Stadler WM, Ahmann FR et al.:

MVA-MUC1-IL2 vaccine immunotherapy (Tg4010) improves PSA doubling time in patients with prostate cancer with biochemical failure. Invest. New Drugs 27(4), 379–386 (2009).

Most recent published result using Transgene’s MVA-MUC1-IL-2 (TG4010) vaccine. Having previously observed efficacy in non-small-cell lung carcinoma, this Phase II study recruited 40 prostate cancer patients, with 13 out of 40 demonstrating at least a twofold increase in prostate-specific antigen doubling time.


structures of two H-2Db/glycopeptide complexes suggest a molecular basis for CTL cross-reactivity. Immunity 10(1), 63–74 (1999). 171 Dutoit V, Rubio-Godoy V, Doucey MA et al.:

172 Vlad AM, Kettel JC, Alajez NM, Carlos CA,

Finn OJ: MUC1 immunobiology: from discovery to clinical applications. Adv. Immunol. 82, 249–293 (2004).

161 Sancho D, Mourão-Sá D, Joffre OP et al.:

Tumor therapy in mice via antigen targeting to a novel, DC-restricted C-type lectin. J. Clin. Invest. 118(6), 2098–2110 (2008).

173 Rudd PM, Elliott T, Cresswell P, Wilson IA,

Dwek RA: Glycosylation and the immune system. Science 291(5512), 2370–2376 (2001).

162 Brandes M, Willimann K, Bioley G et al.:

Cross-presenting human JG T cells induce robust CD8 + DE T cell responses. Proc. Natl Acad. Sci. USA 106(7), 2307–2312 (2009).

174 Xu Y, Gendler SJ, Franco A: Designer

glycopeptides for cytotoxic T cell-based elimination of carcinomas. J. Exp. Med. 199(5), 707–716 (2004).

163 Apostolopoulos V, Pietersz GA, Gordon S,

Martinez-Pomares L, Mckenzie IF: Aldehydemannan antigen complexes target the MHC class I antigen-presentation pathway. Eur J. Immunol. 30(6), 1714–1723 (2000).

153 Johnen H, Kulbe H, Pecher G: Long-term

tumor growth suppression in mice immunized with naked DNA of the human tumor antigen mucin (MUC1). Cancer Immunol. Immunother. 50(7), 356–360 (2001).

et al.: A glycopeptide in complex with MHC class I uses the galnac residue as an anchor. Proc. Natl Acad. Sci. USA 100(25), 15029–15034 (2003).

159 Gulley JL, Arlen PM, Tsang KY et al.: Pilot

151 Grimshaw MJ, Papazisis K, Picco G et al.:

Immunisation with ‘naive’ syngeneic dendritic cells protects mice from tumour challenge. Br. J. Cancer 98(4), 784–791 (2008).

169 Apostolopoulos V, Yuriev E, Ramsland PA

et al.: Poxvirus-based vaccine therapy for patients with advanced pancreatic cancer. J. Transl. Med. 5, 60 (2007).

150 Wang K, Zhou Q, Guo AL, Xu CR, An SJ,

Wu YL: An autologous therapeutic dendritic cell vaccine transfected with total lung carcinoma RNA stimulates cytotoxic T lymphocyte responses against non-small cell lung cancer. Immunol. Invest. 38(7), 665–680 (2009).

PERSPECTIVE

175 Haurum JS, Arsequell G, Lellouch AC et al.:

Recognition of carbohydrate by major histocompatibility complex class I-restricted, glycopeptide-specific cytotoxic T lymphocytes. J. Exp. Med. 180(2), 739–744 (1994).

164 Ninkovic T, Hanisch FG: O-glycosylated

human MUC1 repeats are processed in vitro by immunoproteasomes. J. Immunol. 179(4), 2380–2388 (2007).

First paper to demonstrate the cleavage of MUC1 glycopeptides by immunoproteasomes, beautifully characterizing the effect of glycosylation on different VNTR residues.

165 Ninkovic T, Kinarsky L, Engelmann K et al.:

Identification of O-glycosylated decapeptides within the MUC1 repeat domain as potential MHC class I (A2) binding epitopes. Mol. Immunol. 47(1), 131–140 (2009). 166 Vlad AM, Muller S, Cudic M et al.: Complex

carbohydrates are not removed during processing of glycoproteins by dendritic cells: processing of tumor antigen MUC1 glycopeptides for presentation to major histocompatibility complex class II-restricted T cells. J. Exp. Med. 196(11), 1435–1446 (2002). 167 Haurum JS, Hoier IB, Arsequell G et al.:

Presentation of cytosolic glycosylated peptides by human class I major histocompatibility complex molecules in vivo. J. Exp. Med. 190(1), 145–150 (1999). 168 Stepensky D, Tzehoval E, Vadai E,

Eisenbach L: O-glycosylated versus non-glycosylated MUC1-derived peptides as potential targets for cytotoxic immunotherapy of carcinoma. Clin. Exp. Immunol. 143(1), 139–149 (2006).


176 Tarp MA, Clausen H: Mucin-type

O-glycosylation and its potential use in drug and vaccine development. Biochim. Biophys. Acta 1780(3), 546–563 (2008).

Comprehensive review of MUC1’s O-glycosylation and its implications in immunotherapy. The illustrations help communicate the facts very effectively.

177 Hanada K, Yewdell JW, Yang JC: Immune

recognition of a human renal cancer antigen through post-translational protein splicing. Nature 427(6971), 252–256 (2004). 178 Vigneron N, Ooms A, Morel S, Ma W,

Degiovanni G, Van Den Eynde BJ: A peptide derived from melanocytic protein gp100 and presented by HLA-b35 is recognized by autologous cytolytic T lymphocytes on melanoma cells. Tissue Antigens 65(2), 156–162 (2005). 179 Godefroy E, Moreau-Aubry A, Diez E,

Dreno B, Jotereau F, Guilloux Y: D v E3-dependent cross-presentation of matrix metalloproteinase-2 by melanoma cells gives rise to a new tumor antigen. J. Exp. Med. 202(1), 61–72 (2005). 180 Paludan C, Schmid D, Landthaler M et al.:

Endogenous MHC class II processing of a viral nuclear antigen after autophagy. Science 307(5709), 593–596 (2005).

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PERSPECTIVE


181 Chapiro J, Claverol S, Piette F et al.:

Destructive cleavage of antigenic peptides either by the immunoproteasome or by the standard proteasome results in differential antigen presentation. J. Immunol. 176(2), 1053–1061 (2006).

191 Rabinovich GA, Gabrilovich D,

183 Nencioni A, Grunebach F, Patrone F,

Ballestrero A, Brossart P: Proteasome inhibitors: antitumor effects and beyond. Leukemia 21(1), 30–36 (2007). 184 Nakao M, Sata M, Saitsu H et al.:

CD4 + hepatic cancer-specific cytotoxic T lymphocytes in patients with hepatocellular carcinoma. Cell. Immunol. 177(2), 176–181 (1997). 185 Jerome KR, Barnd DL, Bendt KM et al.:

Cytotoxic T-lymphocytes derived from patients with breast adenocarcinoma recognize an epitope present on the protein core of a mucin molecule preferentially expressed by malignant cells. Cancer Res. 51(11), 2908–2916 (1991). 186 Wright SE, Rewers-Felkins KA, Quinlin IS

et al.: MHC-unrestricted lysis of MUC1expressing cells by human peripheral blood mononuclear cells. Immunol. Invest. 37(3), 215–225 (2008). 187 Magarian-Blander J, Ciborowski P, Hsia S,

Watkins SC, Finn OJ: Intercellular and intracellular events following the MHCunrestricted TCR recognition of a tumorspecific peptide epitope on the epithelial antigen MUC1. J. Immunol. 160(7), 3111–3120 (1998). 188 Alajez NM, Schmielau J, Alter MD, Cascio M,

Finn OJ: Therapeutic potential of a tumorspecific, MHC-unrestricted T-cell receptor expressed on effector cells of the innate and the adaptive immune system through bone marrow transduction and immune reconstitution. Blood 105(12), 4583–4589 (2005). 189 Chen X, Gao W, Gambotto A, Finn OJ:

Lentiviral vectors encoding human MUC1-specific, MHC-unrestricted singlechain TCR and a fusion suicide gene: potential for universal and safe cancer immunotherapy. Cancer Immunol. Immunother. 58(6), 977–987 (2009). 190 Mbeunkui F, Johann DJ Jr: Cancer and the

tumor microenvironment: a review of an essential relationship. Cancer Chemother. Pharmacol. 63(4), 571–582 (2009).

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T cell suppression as a mechanism for tolerance to MUC1 antigen in MUC1 transgenic mice. Breast Cancer Res. Treat. 60(2), 107–115 (2000).

192 Domschke C, Schuetz F, Ge Y et al.:

182 Kuhn DJ, Hunsucker SA, Chen Q,

Voorhees PM, Orlowski M, Orlowski RZ: Targeted inhibition of the immunoproteasome is a potent strategy against models of multiple myeloma that overcomes resistance to conventional drugs and nonspecific proteasome inhibitors. Blood 113(19), 4667–4676 (2009).

202 Chen D, Koido S, Li Y, Gendler S, Gong J:

Sotomayor EM: Immunosuppressive strategies that are mediated by tumor cells. Annu. Rev. Immunol. 25, 267–296 (2007).

Intratumoral cytokines and tumor cell biology determine spontaneous breast cancer-specific immune responses and their correlation to prognosis. Cancer Res. 69(21), 8420–8428 (2009).

203 Agrawal B, Krantz MJ, Reddish MA,

Analysis of the natural immunological response in 207 breast cancer patients. By assessing many factors and with a large patient number, a number of interesting conclusions can be drawn, with the authors stressing the importance of the microenvironment in any response.

204 Tinder TL, Subramani DB, Basu GD et al.:

Longenecker BM: Cancer-associated MUC1 mucin inhibits human T-cell proliferation, which is reversible by IL-2. Nat. Med. 4(1), 43–49 (1998). MUC1 enhances tumor progression and contributes toward immunosuppression in a mouse model of spontaneous pancreatic adenocarcinoma. J. Immunol. 181(5), 3116–3125 (2008). 205 Ilkovitch D, Lopez DM: Urokinase-mediated

recruitment of myeloid-derived suppressor cells and their suppressive mechanisms are blocked by MUC1/sec. Blood 113(19), 4729–4739 (2009).

193 Wright SE, Rewers-Felkins KA, Quinlin IS

et al.: Tumor burden influences cytotoxic T cell development in metastatic breast cancer patients – a Phase I/II study. Immunol. Invest. 38(8), 820–838 (2009).

206 Rubinstein DB, Karmely M, Pichinuk E et al.:

The MUC1 oncoprotein as a functional target: Immunotoxin binding to D/E junction mediates cell killing. Int. J. Cancer 124(1), 46–54 (2009).

194 Zhang K, Sikut R, Hansson GC: A MUC1

mucin secreted from a colon carcinoma cell line inhibits target cell lysis by natural killer cells. Cell. Immunol. 176(2), 158–165 (1997). 195 Ogata S, Maimonis PJ, Itzkowitz SH:

Mucins bearing the cancer-associated sialosyl-Tn antigen mediate inhibition of natural killer cell cytotoxicity. Cancer Res. 52(17), 4741–4746 (1992). 196 Yokoigawa N, Takeuchi N, Toda M et al.:

Enhanced production of interleukin 6 in peripheral blood monocytes stimulated with mucins secreted into the bloodstream. Clin. Cancer Res. 11(17), 6127–6132 (2005). 197 Ishida A, Ohta M, Toda M et al.: Mucin-

induced apoptosis of monocyte-derived dendritic cells during maturation. Proteomics 8(16), 3342–3349 (2008). 198 Mungul A, Cooper L, Brockhausen I et al.:

Sialylated core 1 based O-linked glycans enhance the growth rate of mammary carcinoma cells in MUC1 transgenic mice. Int. J. Oncol. 25(4), 937–943 (2004). 199 Rughetti A, Pellicciotta I, Biffoni M et al.:

Recombinant tumor-associated MUC1 glycoprotein impairs the differentiation and function of dendritic cells. J. Immunol. 174(12), 7764–7772 (2005). 200 Monti P, Leone BE, Zerbi A et al.: Tumor-

derived MUC1 mucins interact with differentiating monocytes and induce IL-10highIL-12low regulatory dendritic cell. J. Immunol. 172(12), 7341–7349 (2004). 201 Uehara F, Ohba N: MUC1 and sialoglycan

expression associated with cytotoxic T lymphocyte infiltration in eyelid malignant tumors. Jpn J. Ophthalmol. 46(3), 237–243 (2002).


First effective use of an antibody to the noncleaved portion of MUC1, demonstrating efficacy in xenotransplant model. Small peptides and other antibodies from other groups are also demonstrating great promise.

207 Mahanta S, Fessler SP, Park J, Bamdad C:

A minimal fragment of MUC1 mediates growth of cancer cells. PLoS One 3(4), E2054 (2008). 208 Hikita ST, Kosik KS, Clegg DO, Bamdad C:

MUC1* mediates the growth of human pluripotent stem cells. PLoS One 3(10), E3312 (2008). 209 Bitler BG, Menzl I, Huerta CL et al.:

Intracellular MUC1 peptides inhibit cancer progression. Clin. Cancer Res. 15(1), 100–109 (2009). 210 Raina D, Ahmad R, Joshi MD et al.: Direct

targeting of the mucin 1 oncoprotein blocks survival and tumorigenicity of human breast carcinoma cells. Cancer Res. 69(12), 5133–5141 (2009). 211 Andre F, Schartz NE, Chaput N et al.:

Tumor-derived exosomes: a new source of tumor rejection antigens. Vaccine 20(Suppl. 4), A28–A31 (2002). 212 Cho JA, Yeo DJ, Son HY et al.: Exosomes:

a new delivery system for tumor antigens in cancer immunotherapy. Int. J. Cancer 114(4), 613–622 (2005). 213 Bozzacco L, Trumpfheller C, Huang Y et al.:

HIV gag protein is efficiently cross-presented when targeted with an antibody towards the DEC-205 receptor in Flt3 ligand-mobilized murine DC. Eur J. Immunol. 40(1), 36–46 (2009).


MUC1 immunotherapy

214 Nencioni A, Brossart P: Crosspresentation:

a matter of pH. Blood 112(12), 4368–4369 (2008). 215 Accapezzato D, Visco V, Francavilla V et al.:

Chloroquine enhances human CD8 + T cell responses against soluble antigens in vivo. J. Exp. Med. 202(6), 817–828 (2005). 216 Ding C, Wang L, Marroquin J, Yan J:

Targeting of antigens to B cells augments antigen-specific T-cell responses and breaks immune tolerance to tumor-associated antigen MUC1. Blood 112(7), 2817–2825 (2008). 217 Zocchi MR, Poggi A: Role of JG

T lymphocytes in tumor defense. Front. Biosci. 9, 2588–2604 (2004). 218 Garnett CT, Palena C, Chakraborty M,

Tsang KY, Schlom J, Hodge JW: Sublethal irradiation of human tumor cells modulates phenotype resulting in enhanced killing by cytotoxic T lymphocytes. Cancer Res. 64(21), 7985–7994 (2004). 219 Gelbard A, Garnett CT, Abrams SI et al.:

Combination chemotherapy and radiation of human squamous cell carcinoma of the head and neck augments CTL-mediated lysis. Clin. Cancer Res. 12(6), 1897–1905 (2006).


220 Andarawewa KL, Paupert J, Pal A,

Barcellos-Hoff MH: New rationales for using TGFE inhibitors in radiotherapy. Int. J. Radiat. Biol. 83(11–12), 803–811 (2007). 221 Zitvogel L, Apetoh L, Ghiringhelli F, Kroemer

G: Immunological aspects of cancer chemotherapy. Nat. Rev. Immunol. 8(1), 59–73 (2008). 222 Mukherjee P, Basu GD, Tinder TL et al.:

Progression of pancreatic adenocarcinoma is significantly impeded with a combination of vaccine and COX-2 inhibition. J. Immunol. 182(1), 216–224 (2009). 223 Moreno M, Mol BM,

Von Mensdorff-Pouilly S et al.: Toll-like receptor agonists and invariant natural killer T-cells enhance antibody-dependent cell-mediated cytotoxicity (ADCC). Cancer Lett. 272(1), 70–76 (2008). 224 Yuan S, Shi C, Han W, Ling R, Li N,

Wang T: Effective anti-tumor responses induced by recombinant bacillus Calmette–Guerin vaccines based on different tandem repeats of MUC1 and GM-CSF. Eur. J. Cancer Prev. 18(5), 416–423 (2009).


PERSPECTIVE

225 Engelmann K, Shen H, Finn OJ: MCF7 side

population cells with characteristics of cancer stem/progenitor cells express the tumor antigen MUC1. Cancer Res. 68(7), 2419–2426 (2008).

 Websites 301 Registry of federally and privately supported

clinical trials. www.clinicaltrials.gov 302 Discontinued Phase II breast cancer trial of

AS1402 (humanized HMFG1) in combination with letrozole. www.antisoma.com/asm/media/press/ pr2009/2009–2008–07 303 Homepage of the biopharmaceutical

company, Transgene. www.transgene.fr 304 News report of the Results of the Phase III

PANVAC-VF trial and Therion’s announcement of liquidation. www.medicalnewstoday.com/articles/46137. php

327