Latest developments in MUC1 immunotherapy.

Background

MUC1 was the first membrane mucin to be identified and the first mucin to be characterized structurally [ 1 – 4 ] (see Figures 1 and 2 ). The protein is cleaved during translation [ 5 , 6 ], and the tandem repeat (TR) containing external domain (25–100 TRs) is bound to the membrane indirectly by non-covalently interacting with the MUC1-C domain that consists of a short extracellular domain (EC) and the transmembrane and cytoplasmic domains (see Figure 2 ). MUC1-C is known to be involved in signal transduction [ 7 , 8 ]. Figure 2. The structure of the MUC1 mucin. During translation, MUC1 is cleaved into two domains, MUC1-N and MUC1-C. MUC1-N consists predominantly of the TR domain and the sequence of a single repeat is illustrated. The TR domain is glycosylated with O-linked glycans (in red) and each repeat has five potential sites shown in bold. There are also sites for O-linked glycosylation in the degenerate TRs located to the N- and C-termini of the repeats. There are five potential sites for N-linked glycosylation close to the membrane (in black). MUC1-C consists of 58 amino acids of the external domain, the transmembrane domain (in blue, 28 aa) and the cytoplasmic domain (MUC1-CD, 72 aa). Within the CD1 domain, the CQC trimer is responsible for homodimerization. There are many phosphorylation sites within the CD domain and two of these are indicated. The CQC containing peptide (GO-232) targets the homodimerization domain.

The MUC1 glycoprotein was first identified as the antigen found in extracts of the human milk fat globule membranes, or in extracts of cancer cells, which induced a strong humoral response in mice, leading to the production of many monoclonal antibodies reactive with the TR domain [ 9 – 12 ]. These antibodies were used to clone the MUC1 gene and differences in the reactivity of some antibodies with normal epithelia and carcinomas were observed [ 13 , 14 ]. MUC1 is extensively glycosylated and the differences in antibody reactivity were found to be due to changes in the O-glycosylation of the serines and threonines in the TR domains of the cancer mucin where the glycans added were truncated and more heavily sialylated than in the normal epithelia [ 15 , 16 ] (see Figure 3 ). Figure 3. Simplified pathways of mucin-type O-linked glycosylation in normal and malignant breast epithelial cells. The Tn glycan can be carried on MUC1 expressed by a high proportion of breast cancers. The STn glycan is found in ∼25% of breast cancers, while ST is more commonly expressed. The unsialylated core 1 (T) is also widely found on MUC1 expressed by breast cancers. In contrast, the glycans found on MUC1 expressed by normal mammary epithelial cells are core 2 based.

Many of the MUC1-reactive antibodies reacted with peptide sequences in the TR domain, and the concept arose that in the cancer mucin, with shorter glycan chains, some of these peptide sequences were more exposed, thus explaining the tumour specificity of antibodies such as SM3 and DF3-P [ 17 , 18 ]. This led to the belief that peptide antigens based on the MUC1 TR sequences would induce tumour-specific adaptive immune responses. Many small Phase I/Phase II clinical trials using MUC1-based peptide immunogens were initiated, but these have not graduated to larger Phase III trials [ 19 ]. This may reflect the paucity of MHC class I epitopes within the TR of MUC1. Nevertheless, four MUC1-based Phase III trials were performed, one of which used an MUC1 peptide as immunogen (L-BLP25) [ 20 ]. These trials were completed several years ago and the therapies investigated, outlined in Table 1 , are not under further evaluation (see Outcomes and Comments in Table 1 ). Table 1 Phase III MUC1-based clinical trials Vaccine Cancer/number of patients Treatment Outcome Comment Investigators/year Peptide vaccine: L-BLP25 (tecemotide) Lyophilized 25mer MUC1 TR lipopeptide + lipid adjuvant N = 1513 Stage IIIb NSCLC patients Administered post chemoradiotherapy versus placebo. Cyclophosphamide administered once before first tecemotide administration, or saline prior to placebo No overall survival (OS) difference or time to relapse within whole cohort. OS significantly increased in subgroup treated after simultaneous chemo- and radiotherapy with L-BLP25 ( n = 538) versus placebo ( n = 238): 30.6 versus 20.6 months ( P = 0.016) Claimed that MUC1-specific cytotoxic T cells were induced; however, Class I epitopes for CD8 stimulation are found outside TR. Control group with MUC1 liposomes not included therefore unclear of contribution of lipid stimulation of Toll receptor versus MUC1 effects. [ 20 ] Glycan vaccine: Sialyl Tn-KLH N = 1028 Breast cancer patients Sialyl Tn-KLH versus KLH alone. Cyclophosphmide included in all treatments to down-regulate Tregs No significant difference in OS in patients receiving sialyl Tn-KLH versus KLH alone Only 20–25% of breast tumours express sialyl Tn and expression of STn on patients tumours was not assessed. Still unclear whether vaccine could be effective in patients with STn-positive cancers [ 62 ] Viral vaccine: PANVAC-VF viral vector expressing CEA and MUC1 plus B7.1, intracellular adhesion molecule-1 and leukocyte function-associated antigen-3 N = 255 Advanced pancreatic cancer patients PANVAC-VF versus palliative chemotherapy No significant difference in OS of patients receiving PANVAC-VF versus palliative chemotherapy or best supportive care Plans to file Biologics License Application (BLA) with FDA abandoned Therion Unpublished www.medicalnewstoday.com/releases/46137 June 2006 Antibody vaccine: murine MAb HMFG1 carrying Yttrium-90 N = 447 ovarian cancer patients in remission after surgery and chemotherapy Single dose 90 Y HMFG1 versus placebo No OS or time to relapse seen in antibody-treated group compared with placebo HMFG1 does not show good specificity for cancer-associated mucin. Use of a murine Ab is not appropriate. HMFG1 was subsequently humanized and retried in phase II (AS1402 and BTH1704) before being discontinued [ 63 ] Abbreviations: TR, tandem repeat; KLH, keyhole limpet haemocyanin; MAb, monoclonal antibody; OS, overall survival; STn, sialyl Tn; CEA, carcinoembryonic antigen.

Phase I/II trials reported in the literature

The viral vaccine TG4010

The viral vaccine TG4010 developed by Transgene has shown promising results and is moving to further trials. TG4010 is a modified Vaccinia Ankara strain expressing a full-length MUC1 (containing five TRs) and IL-2. The Phase IIb trial in non-small cell lung cancer (NSCLC) patients was completed, where the viral vaccine was administered in combination with chemotherapy [ 21 ]. Response to the viral vaccine was predicted by levels of natural killer (NK) cells and TG4010 plus chemotherapy improved progression-free survival compared with chemotherapy and placebo. Further studies on the patients and tumours from this trial indicate that TG4104 induces a broadening of the T-cell response to other tumour-associated antigens and the diversity of the T-cell response relates to response to therapy [ 22 ].

With FDA approval, a Phase II clinical trial has been initiated for the first-line treatment of NSCLC patients, exploring the effect of combining TG4010 with chemotherapy and the Bristol-Myers Squibb's immune checkpoint inhibitor nivolumab [ 23 ]. This study is now recruiting participants.

Comment : After injection of the virus, full-length MUC1 glycoprotein is expressed; therefore, Class I epitopes outside of the TR domain can be presented. Moreover, the epitope spreading to other TAA is a positive parameter. Combining the vaccine with the checkpoint inhibitor could also enhance efficacy.

Several Phase I/II trials injecting autologous dendritic cell

Several Phase I/II trials injecting autologous dendritic cell (AuDC) loaded with MUC1 as a peptide, mRNA or fused to tumour cells have been reported [ 24 – 31 ]. The time taken for the preclinical data using dendritic cells (DCs) fused to tumour cells to be translated to the clinic and show efficacy [ 28 , 29 ] illustrates the length of time required for incorporating research into the clinic.

The trials using AuDCs loaded with oxidized mannan linked to a 5TRMUC1 peptide also show promise. Unusually, this trial in breast cancer patients has a 15-year follow-up [ 32 ] when the recurrence rate in patients receiving placebo was 60% (nine of 15) compared with 12.5% (two of 16) for those receiving immunotherapy.

Comment for the oxidized mannan trial: This immunogen is now called CVac by the company Prima Biomed, which is no longer recruiting patients but is seeking commercial partners for the product.

Adoptive immunotherapy with Gemcitabine

The investigators of the present study specify that it was not a trial but a medical treatment approved as advanced health care by the Japanese Ministry of Health, Labor and Welfare, and patients were self-funded. Forty-two late-stage pancreatic patients were given injections of autologous DCs pulsed with full-length MUC1 mRNA and autologous CTLs generated in vitro. Average mean survival time (MST) for all patients was 13.9 months, and the 1-year survival rate was 51.1%. For patients ( n = 30) who received more than 1 × 10 7 MUC1-DCs per injection and 3 × 10 8 MUC1-CTLs per injection, MST was 16.5 months versus MST of 5.7 months in the other 12 patients ( P = 0.00020) [ 33 ].

Comment: CTLs were generated using one cell line — YPK-1 (HLA-A2402) — assuming the generation of Class I non-restricted MUC1-reactive CTLs. Such CTLs have been reported [ 34 ], but they have proved to be difficult to characterize. Nevertheless, the study documents effective immune responses to MUC1.

Preclinical studies and combination therapies

Preclinical studies continue to be initiated using various vaccine formats of MUC1 based on the glycosylated or unglycosylated TR sequence. Investigators in the Kunz laboratory have chemically synthesized a wide range of glycopeptides based on the TR of MUC1 and have evaluated various formulations of these (containing factors to recruit innate immunity and/or CD4 help) for antibody induction in WT mice [ 35 , 36 ]. Some of these new antibodies show specificity for the tumour MUC1 glycoforms [ 37 ] and humoral and cellular immune responses to one vaccine formulation (1TR carrying Tn and STn bound to tetanus toxin) have been detected in MUC1 transgenic mice where human MUC1 is expressed from the MUC1 promoter [ 38 ].

A study using MUC1 transgenic mice for evaluation of the effect of an anti-PD-L1 antibody on the growth of an aggressive MUC1 expressing murine ovarian cancer cell line (2F8) is illuminating [ 39 ]. Tumour-bearing MUC1 transgenic mice treated with anti-PD-L1 antibody (21-day post tumour injection) showed increased survival and T-cell infiltration within the tumour, but induction of MUC1-specific antibodies was low.

Combination immunotherapy (using checkpoint inhibitors together with MUC1 strategies) is now being initiated as well as testing MUC1 as an immunogen together with other antigens ( https://clinicaltrials.gov/ ), but data are not yet available. If several antigens are presented, interpretation regarding the contribution of MUC1 to effects on tumour growth, which may be documented, will be difficult.

Applying research findings to immunotherapeutic strategies

Apart from the developments on immune checkpoint inhibitors, investigations into the interactions of cancers with the immune system and with their local environment are leading to other important findings. Information relating to the importance of the change in glycosylation of MUC1 and the interaction of specific cancer MUC1 glycoforms with cells in the microenvironment are also suggesting novel approaches. Moreover, a role for the MUC1-C component is becoming of central importance.

Importance of aberrant glycosylation of MUC1 in immunotherapy

A role for glycopeptide epitopes in inducing not only humoral but also cellular adaptive immune responses has been recognized for many years [ 40 ], but it is only relatively recently that the focus on glycosylation changes has been seriously pursued in the search for effective MUC1-related immunotherapy.

Antibodies to glycopeptide epitopes in the TR domain

PankoMab-GEX

Many MUC1-specific antibodies react with epitopes within the PDTRP sequence and reactivity is affected by glycosylation [ 11 , 17 , 18 , 41 ]. The PankoMab antibody reacts with a conformational epitope where the threonine in PDTRP carries the Tn or T glycan and selectively reacts with the cancer mucin [ 42 ]. PankoMab-GEX has been humanized and glyco-optimized for improved ADCC and ADCP activity and enhanced NK cell killing. In a Phase I trial [ 43 ] ( NCT01222624 ), 74 patients with advanced MUC1-positive carcinomas received PankoMab-GEX intravenously in a three-plus-three dose-escalation design until disease progression. PankoMab-GEX showed promising anti-tumour activity in advanced disease and a Phase IIb study is now ongoing evaluating the efficacy of PankoMab-GEX as maintenance therapy in advanced ovarian cancer.

Retargeting of human T cells to tumour-associated MUC1 glycoforms

Chimeric antigen receptors (CARs) targeting T cells to MUC1 were first developed and characterized by the Maher group [ 44 ] using the SM3 antibody, which is highly selective for cancer MUC1 glycoforms [ 17 ], and the HMFG2 antibody, which also shows some selectivity. Several CARs were engineered and the HMFG2 CAR containing a fused CD28 plus OX40 plus CD3 endodomain (HOX HMFG2) together with the IgD linker, to combat glycosylation-independent steric hindrance, was shown to inhibit breast cancer growth in a mouse model.

More recently, a CAR fusion protein with a humanized version of the 5E5 antibody has been developed [ 45 ] based on the evidence that 5E5 shows impressive specificity in reacting selectively with the Cancer MUC1Tn glycoform. 5E5 was raised against a 60mer TR peptide carrying five GalNAc residues (Tn) per TR and reacts preferentially with the GSTA sequence carrying Tn and to a lesser extent STn [ 46 , 47 ]. Unlike PankaMab, which recognizes the PDTR epitope carrying either Tn or T, 5E5 does not recognize the GSTA epitope carrying T.

Studies with these CAR constructs have not yet advanced to the clinic: MUC1 targeting CAR cells have been held in the preclinical setting for many years while technical and biological challenges are overcome — not least the expression of MUC1 on T cells themselves [ 48 ]. The specificity of the 5E5 CAR, which does not bind the glycoform of MUC1 on T cells [ 49 ], could circumvent problems that might be encountered with other MUC1 CARs, which do bind to MUC1 on T cells.

There are 10 active Phase I/II CAR trials targeting MUC1 in multiple solid and non-solid malignancies currently recruiting: nine using α/β T cells and one using NK cells, some of which are trialling combination therapy with checkpoint blockade ( https://clinicaltrials.gov/ ). Identification of the antibodies, from which the CARs were derived, is not always easy and the trials are in regions where the stringency of requirements for cell therapy is not as high as in Europe or the USA. Nevertheless, it will be interesting to see how these trials develop.

CD4 T-cell responses to MUC1 glycopeptides

When presented by DCs in MUC1 transgenic mice, MUC- reactive CD4 T-cell responses have been documented [ 28 , 50 ], along with the induction of humoral immunity [ 28 , 50 ] . Moreover, several investigators have demonstrated that MUC1 vaccines carrying the Tn and/or STn glycan are not subject to self-tolerance in MUC1 transgenic mice [ 38 , 46 , 51 ].

These observations are now being taken further along the road to clinical evaluation and a small Phase I clinical trial has been reported where a DC vaccine loaded with 100mer TR MUC1 peptide carrying Tn was administered with KLH to non-metastatic, castrate-resistant prostate cancer patients [ 52 ]. This trial was preceded by a study in Rhesus Macaques where the corresponding Tn-rmMUC1 in autologous DCs was administered with an adjuvant and induction of antibodies and T-cell activation monitored. The rmMUC1 study demonstrated T-cell activation but MUC1-reactive antibodies were not induced. The same profile of activation of the adaptive immune response was seen in the prostate cancer patients: predominantly, CD4 T-cell responses were seen with CD8+ responses being variable, while no MUC1-reactive antibodies were induced.

Eleven of 16 patients treated showed an increase in PSA doubling time (10.5 months) compared with the pre-vaccination value (4.6 months) .

MUC1 effects on the tumour environment and immune effector cells

It is becoming apparent that MUC1 expressed by cancer cells can affect the phenotype and function of immune cells in the tumour microenvironment inhibiting their function.

Effects of a cancer glycoform of MUC1 on myeloid-derived cells

• The dominant glycoform of MUC1 expressed by most carcinomas is MUC1-ST (see Figure 3 ), which is expressed on the cell surface and can be secreted. Beatson et al. have shown that MUC1-ST interacts with the sialic acid-binding protein Siglec-9 expressed on monocytes and macrophages: this interaction induces the development of tumour-associated macrophages showing increased expression of the PD-L1 checkpoint ligand as well as other proteins associated with tumour growth and metastases [ 53 ]. Many proteins of the Siglec family of sialic acid-binding proteins are involved in immune suppression of innate and adaptive immune effector cells and tumours exploit these ‘Glycoimmune checkpoints’. The Palleon Pharmaceutical Company is developing antibodies to block the glycan–Siglec interactions, including that of Siglec 9 with MUC1-ST ( Palleonpharma.com ).

• Induction of myeloid-derived suppressor cells ( MDSCs ) by MUC1 in acute myeloblastic/myelogenous leukaemia (AML) cells has also been reported [ 54 ]. MDSCs, which derive from immature monocytes and suppress effector T cells, were induced by extracellular vesicles from AML cells. MUC1 is a critical component of these vesicles and up-regulates c-MYC expression by down-regulating the expression of MiR34 that targets c-MYC. By silencing expression of MUC1, c-MYC target genes are suppressed in the recipient myeloid cells and levels of MDSCs are significantly reduced. Although not specifically shown to be the domain of MUC1 responsible for the generation of MDSCs, the MUC1-C cytoplasmic component is a candidate. Moreover, this component of MUC1 has been shown to contribute to immune evasion in other ways ([ 55 ] and see below).

Targeting the transmembrane component of MUC1 (MUC1-C)

Although much of the work relating to MUC1 as a therapeutic target in cancer has focused on the EC domain, interest in the smaller membrane spanning component (MUC1-C) has increased.

Preclinical studies showed that MUC1-C interacts with EGFR and other membrane receptors and that the cytoplasmic domain is transported to the nucleus where it activates multiple signalling pathways [ 7 ]. Consequently, a peptide inhibitor (GO-203) has been developed and directed to the CQC sequence in the cytoplasmic domain of MUC1-C, which is required for dimerization and signalling activity ( Figure 2 ). Using CRISPR gene editing and/or the GO-203 inhibitor to silence MUC1 in multiple myeloma cells, MUC1-C was shown to activate MYC expression by binding to the promoter in a β-catenin/transcription factor 4-mediated mechanism [ 56 ]. Activation of MYC leads to increased expression of MYC targets including BMI and other components of the Polycomb 1 complex [ 57 ].

Clearly, these data indicate that the molecular mechanisms activating expression of MYC are different in cells derived from different tumours (see MUC1, MYC and MDSCs above). This also applies to the mechanisms reported to underlie the induction of the PD-L1 ligand by MUC1 in different tumour types. In NSCLC cell lines, MUC1-C is associated with NF-κBp65 at the promoter of PD-L1 thus directly affecting transcription and is also associated with immune evasion and a suppressive immune microenvironment [ 58 , 59 ]. On the other hand, in AML cells, MUC1 is reported to inhibit the expression of several microRNAs targeting PD-L1 by inhibiting Dicer [ 60 ]. Nevertheless, effects of MUC1-C on PD-L1 induction and immune invasion are widely seen in other cancers, including triple-negative breast cancer [ 61 ], and the MUC1-C inhibitor GO-203 has now completed a Phase I trial in patients with advanced solid tumours: patients are now being recruited for a Phase II trial of GO-203 in combination with decitabine for patients with AML ( NCT02204085 ).

Summary

Efforts continue to investigate strategies for exploiting the overexpression and aberrant glycosylation of MUC1 in multiple cancer types and possible approaches to MUC1-based immunotherapy are summarized in Figure 4 . Some recently initiated clinical trials are taking on board the novel findings emerging from work on the structure and function of MUC1 and its interaction with the tumour environment and immune effector cells. These data indicate that both the MUC1-N (highly glycosylated) and MUC1-C components contribute to immune evasion by cancer cells, and this could be an important consideration in the strategic development of MUC1-based immunotherapy. The findings emphasize that although testing the in vitro findings in clinical trials is important, it is also important to continue preclinical studies aimed at further defining the function of this glycoprotein in cancer. Figure 4. Current and potential clinical approaches targeting MUC1 in cancer. Current strategies fall into six broad categories (as shown in the boxes) ranging from preclinical to Phase III. The bullet point within each box gives an example of that category of immunotherapy. To date, no therapeutic targeting MUC1 has been approved for general use. GM, genetically modified; DC, dendritic cell; APC, antigen-presenting cell; NK, natural killer.

Abbreviations

antibody-dependent cellular cytotoxicity

antibody-dependent cellular phagocytosis

acute myeloblastic/myelogenous leukaemia

autologous dendritic cell

chimeric antigen receptors

myeloid-derived suppressor cells

dendritic cells

extracellular domain

epidermal growth factor receptor

keyhole limpet hemocyanin

mean survival time

mucin 1

natural killer

non-small cell lung cancer

tandem repeat

wild type

Authors Contribution

J.T.-P. prepared the first draft of the manuscript and decided with R.B. and J.B. on the content. J.B. designed and prepared the figures and R.B. recorded the range of trials initiated and ongoing. R.G. produced the table for Phase III trials.

Funding

R.B. was funded through an Medical Research Council grant [MR/R000026/1].

Competing Interests

J.B. is a consultant for Palleon Pharmaceuticals. All other authors declare no conflicts of interest.

References

1 Gendler S.J. , Burchell J.M. , Duhig T. , Lamport D. , White R. , Parker M. et al. ( 1987 ) Cloning a cDNA coding for differentiation and tumour-associated mucin glycoprotein expressed by human mammary epithelium . Proc. Natl Acad. Sci. U.S.A. 84 , 6060 – 6064 10.1073/pnas.84.17.6060 2888110 PMC299007

2 Lan M.S. , Batra S.K. , Qi W.N. , Metzgar R.S. and Hollingsworth M.A. ( 1990 ) Cloning and sequencing of a human pancreatic tumor mucin cDNA . J. Biol. Chem. 265 , 15294 – 15299 PMID: 2394722

3 Ligtenberg M.J. , Vos H.L. , Gennissen A.M. and Hilkens J. ( 1990 ) Episialin, a carcinoma-associated mucin, is generated by a polymorphic gene encoding splice variants with alternative amino termini . J. Biol. Chem. 265 , 5573 – 5578 PMID: 2318825

4 Siddiqui J. , Abe M. , Hayes D. , Shani E. , Yunis E. and Kufe D. ( 1988 ) Isolation and sequencing of a cDNA coding for the human DF3 breast carcinoma-associated antigen . Proc. Natl Acad. Sci. U.S.A. 85 , 2320 – 2323 10.1073/pnas.85.7.2320 2895474 PMC279983

5 Baruch A. , Hartmann M. , Yoeli M. , Adereth Y. , Greenstein S. , Stadler Y. et al. ( 1999 ) The breast cancer-associated MUC1 gene generates both a receptor and its cognate binding protein . Cancer Res. 59 , 1552 – 1561 PMID: 10197628

6 Julian, J. , Dharmaraj, N. and Carson, D.D. ( 2009 ) MUC1 is a substrate for γ-secretase . J. Cell Biochem. 108 , 802 – 815 10.1002/jcb.22292 19711367 PMC3228317

7 Kufe, D.W. ( 2013 ) MUC1-C oncoprotein as a target in breast cancer: activation of signaling pathways and therapeutic approaches . Oncogene 32 , 1073 – 1081 10.1038/onc.2012.158 22580612 PMC3621754

8 Kufe, D.W. ( 2009 ) Mucins in cancer: function, prognosis and therapy . Nat. Rev. Cancer 9 , 874 – 885 10.1038/nrc2761 19935676 PMC2951677

9 Taylor-Papadimitriou J. , Peterson J.A. , Arklie J. , Burchell J. , Ceriani R.L. and Bodmer W.F. ( 1981 ) Monoclonal antibodies to epithelium-specific components of the human milk fat globule membrane: production and reaction with cells in culture . Int. J. Cancer 28 , 17 – 21 10.1002/ijc.2910280104 7309278

10 Hilkens, J. , Buijs, F. , Hilgers, J. , Hageman, P. , Calafat, J. , Sonnenberg, A. et al. ( 1984 ) Monoclonal antibodies against human milk-fat globule membranes detecting differentiation antigens of the mammary gland and its tumors . Int. J. Cancer 34 , 197 – 206 10.1002/ijc.2910340210 6206003

11 Taylor-Papadimitriou J. ( 1991 ) Report on the first international workshop on carcinoma-associated mucins . Int. J. Cancer 49 , 1 – 5 10.1002/ijc.2910490102 1714883

12 Rye P.D. and Price M.R. ( 1998 ) ISOBM TD-4 International workshop on monoclonal antibodies against MUC1 . Workshop, San Diego, CA, November 1996 ISBN: 978-3-8055-6597-4 9441368

13 Arklie J. , Taylor-Papadimitriou J. , Bodmer W.F. , Egan M. and Millis R. ( 1981 ) Differentiation antigens expressed by epithelial cells in the lactating breast are also detectable in breast cancers . Int. J. Cancer 28 , 23 – 29 10.1002/ijc.2910280105 6273328

14 Kufe, D. , Inghirami, G. , Abe, M. , Hayes, D. , Justi-Wheeler, H. and Schlom, J. ( 1984 ) Differential reactivity of a novel monoclonal antibody (DF3) with human malignant versus benign breast tumors . Hybridoma 3 , 223 – 232 10.1089/hyb.1984.3.223 6094338

15 Lloyd K.O. , Burchell J. , Kudryashov V. , Yin B.W.T. and Taylor-Papadimitriou J. ( 1996 ) Comparison of O -linked carbohydrate chains in MUC-1 mucin from normal breast epithelial cell lines and breast carcinoma cell lines: demonstration of simpler and fewer glycan chains in tumor cells . J. Biol. Chem. 271 , 33325 – 33334 10.1074/jbc.271.52.33325 8969192

16 Bäckström M. , Link T. , Olson F.J. , Karlsson H. , Graham R. , Picco G. et al. ( 2003 ) Recombinant MUC1 mucin with a breast cancer-like-O-glycosylation produced in large amounts of Chinese-hamster ovary cells . Biochem. J. 376 , 677 – 686 10.1042/bj20031130 12950230 PMC1223800

17 Burchell J. , Gendler S. , Taylor-Papadimitriou J. , Girling A. , Lewis A. , Millis R. et al. ( 1987 ) Development and characterisation of breast cancer reactive monoclonal antibodies directed to the core protein of the human milk mucin . Cancer Res. 47 , 5476 – 5482 PMID: 2443241

18 Perey, L. , Hayes, D.F. , Maimonis, P. , Abe, M. , O'Hara, C. and Kufe, D.W. ( 1992 ) Tumor selective reactivity of a monoclonal antibody prepared against a recombinant peptide derived from the DF3 human breast carcinoma-associated antigen . Cancer Res. 52 , 2563 – 2568 PMID: 1373671

19 Rivalland, G. , Loveland, B. and Mitchell, P. ( 2015 ) Update on Mucin-1 immunotherapy in cancer: a clinical perspective . Expert Opin. Biol. Ther. 15 , 1773 – 1787 10.1517/14712598.2015.1088519 26453294

20 Butts, C. , Socinski, M.A. , Mitchell, P.L. , Thatcher, N. , Havel, L. , Krzakowski, M. et al. ( 2014 ) Tecemotide (L-BLP25) versus placebo after chemoradiotherapy for stage III non-small-cell lung cancer (START): a randomised, double-blind, phase 3 trial . Lancet Oncol. 15 , 59 – 68 10.1016/S1470-2045(13)70510-2 24331154

21 Quoix, E. , Lena, H. , Losonczy, G. , Forget, F. , Chouaid, C. , Papai, Z. et al. ( 2016 ) TG4010 immunotherapy and first-line chemotherapy for advanced non-small-cell lung cancer (TIME): results from the phase 2b part of a randomised, double-blind, placebo-controlled, phase 2b/3 trial . Lancet Oncol. 17 , 212 – 223 10.1016/S1470-2045(15)00483-0 26727163

22 Torch, C. , Bastien, B. , Barraud, L. , Grellier, B. , Nourtier, V. , Gantzer, M. et al. ( 2017 ) Viral based vaccine TG4010 induces broadening of specific immune response and improves outcome in advanced NSCLC . J. Immunother. Cancer 5 , 70 10.1186/s40425-017-0274-x 28923084 PMC5604422

23 https://clinicaltrials.gov/ct2/show/NCT02823990

24 Mitchell, P.L. , Quinn, M.A. , Grant, P.T. , Allen, D.G. , Jobling, T.W. , White, S.C. et al. ( 2014 ) A phase 2, single-arm study of an autologous dendritic cell treatment against mucin 1 in patients with advanced epithelial ovarian cancer . J. Immunother. Cancer 2 , 16 10.1186/2051-1426-2-16 24995129 PMC4080759

25 Gray, H.J. and Gargosky, S.E. ( 2014 ) CAN-003 Study Team . Progression-free survival in ovarian cancer patients in second remission with mucin-1 autologous dendritic cell therapy . J. Clin. Oncol. 32 ( 15 Suppl ), 5504 10.1200/jco.2014.32.15_suppl.5504

26 Lepisto, A.J. , Moser, A.J. , Zeh, H. , Lee K. , Bartlett D. , McKolanis J.R. et al ( 2008 ) A phase I/II study of a MUC1 peptide pulsed autologous dendritic cell vaccine as adjuvant therapy in patients with resected pancreatic and biliary tumors . Cancer Ther. 6 , 955 – 964 PMID: 19129927 PMC2614325

27 Wierecky, J. , Müller, M.R. , Wirths, S. , Halder-Oehler E. , Dörfel D. , Schmidt S.M. et al ( 2006 ) Immunologic and clinical responses after vaccinations with peptide-pulsed dendritic cells in metastatic renal cancer patients . Cancer Res. 66 , 5910 – 5918 10.1158/0008-5472.CAN-05-3905 16740731

28 Gong, J. , Chen, D. , Kashiwaba, M. , Li, Y. , Chen, L. , Takeuchi, H. et al. ( 1998 ) Reversal of tolerance to human MUC1 antigen in MUC1 transgenic mice immunized with fusions of dendritic and carcinoma cells . Proc. Natl Acad. Sci. U.S.A. 95 , 6279 – 6283 . Point 2 Insert in textp3 under Phase1/II trials DCs 10.1073/pnas.95.11.6279 9600956 PMC27657

29 Rosenblatt, J. , Stone, R.M. , Uhl, L. , Neuberg, D. , Joyce, R. , Levine, J.D. et al. ( 2016 ) Individualized vaccination of AML patients in remission is associated with induction of antileukemia immunity and prolonged remissions . Sci. Transl. Med. 8 , 368ra171 10.1126/scitranslmed.aag1298 PMC5800949 27928025

30 Rittig, S.M. , Haentschel, M. , Weimer, K.J. , Heine A. , Muller M.R. , Brugger W. et al ( 2011 ) Intradermal vaccinations with RNA coding for TAA generate CD8+ and CD4+ immune responses and induce clinical benefit in vaccinated patients . Mol. Ther. 19 , 990 – 999 10.1038/mt.2010.289 21189474 PMC3098631

31 Morse, M.A. , Niedzwiecki, D. , Marshall, J.L. , Garrett C. , Chang D.Z. , Aklilu M. et al ( 2013 ) A randomized phase II study of immunization with dendritic cells modified with poxvectors encoding CEA and MUC1 compared with the same poxvectors plus GM-CSF for resected metastatic colorectal cancer . Ann. Surg. 258 , 879 – 886 10.1097/SLA.0b013e318292919e 23657083 PMC3812363

32 Vassilaros, S. , Tsibanis, A. , Tsikkinis, A. , Pietersz, G.A. , McKenzie, I.F. and Apostolopoulos, V. ( 2013 ) Up to 15-year clinical follow-up of a pilot Phase III immunotherapy study in stage II breast cancer patients using oxidized mannan–MUC1 . Immunotherapy 5 , 1177 – 1182 10.2217/imt.13.126 24188672

33 Shindo, Y. , Hazama, S. , Maeda, Y. , Matsui, H. , Iida, M. , Suzuki, N. et al. ( 2014 ) Adoptive immunotherapy with MUC1-mRNA transfected dendritic cells and cytotoxic lymphocytes plus gemcitabine for unresectable pancreatic cancer . J. Transl. Med. 12 , 175 10.1186/1479-5876-12-175 24947606 PMC4074851

34 Jerome, K.R. , Barnd, D.L. , Bendt, K.M. , Boyer, C.M. , Taylor-Papadimitriou, J. , McKenzie, I.F. et al. ( 1991 ) 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 , 2908 – 2916 PMID: 1709586

35 Gaidzik, N. , Westerlind, U. and Kunz, H. ( 2013 ) The development of synthetic antitumour vaccines from mucin glycopeptide antigens . Chem. Soc. Rev. 42 , 4421 – 4442 10.1039/c3cs35470a 23440054

36 Kaiser, A. , Gaidzik, N. , Becker, T. , Menge, C. , Groh, K. , Cai, H. et al. ( 2010 ) Fully synthetic vaccines consisting of tumor-associated MUC1 glycopeptides and a lipopeptide ligand of the Toll-like receptor 2 . Angew. Chem. Int. Ed. Engl. 49 , 3688 – 3692 10.1002/anie.201000462 20449837

37 Palitzsch, B. , Gaidzik, N. , Stergiou, N. , Stahn, S. , Hartmann, S. , Gerlitzki, B. et al. ( 2016 ) A synthetic glycopeptide vaccine for the induction of a monoclonal antibody that differentiates between normal and tumor mammary cells and enables the diagnosis of human pancreatic cancer . Angew. Chem. Int. Ed. Engl. 55 , 2894 – 2898 10.1002/anie.201509935 26800384

38 Stergiou, N. , Glaffig, M. , Jonuleit, H. , Schmitt, E. and Kunz, H. ( 2017 ) Immunization with a synthetic human MUC1 glycopeptide vaccine against tumor-associated MUC1 breaks tolerance in human MUC1 transgenic mice . ChemMedChem. 12 , 1424 – 1428 10.1002/cmdc.201700387 28675699

39 Mony, J.T. , Zhang, L. , Ma, T. , Grabosch, S. , Tirodkar, T.S. , Brozick, J. et al. ( 2015 ) Anti-PD-L1 prolongs survival and triggers T cell but not humoral anti-tumor immune responses in a human MUC1-expressing preclinical ovarian cancer model . Cancer Immunol. Immunother . 64 , 1095 – 1108 10.1007/s00262-015-1712-6 25998800 PMC4545381

40 Haurum, J.S. , Arsequell, G. , Lellouch, A.C. , Wong, S.Y. , Dwek, R.A. , McMichael, A.J. et al. ( 1994 ) Recognition of carbohydrate by major histocompatibility complex class I-restricted, glycopeptide-specific cytotoxic T lymphocytes . J. Exp. Med. 180 , 739 – 744 10.1084/jem.180.2.739 8046349 PMC2191607

41 Karsten, U. , Serttas, N. , Paulsen, H. , Danielczyk, A. and Goletz, S. ( 2004 ) Binding patterns of DTR-specific antibodies reveal a glycosylation-conditioned tumor-specific epitope of the epithelial mucin (MUC1) . Glycobiology 14 , 681 – 692 10.1093/glycob/cwh090 15115750

42 Danielczyk, A. , Stahn, R. , Faulstich, D. , Löffler, A. , Märten, A. , Karsten, U. et al. ( 2006 ) Pankomab: a potent new generation anti-tumour MUC1 antibody . Cancer Immunol. Immunother. 55 , 1337 – 1347 10.1007/s00262-006-0135-9 16485130 PMC11029825

43 Fiedler, W. , DeDosso, S. , Cresta, S. , Weidmann, J. , Tessari, A. , Salzberg, M. et al. ( 2016 ) A phase I study of PankoMab-GEX, a humanised glyco-optimised monoclonal antibody to a novel tumour-specific MUC1 glycopeptide epitope in patients with advanced carcinomas . Eur. J. Cancer 63 , 55 – 63 10.1016/j.ejca.2016.05.003 27285281

44 Wilkie, S. , Picco, G. , Foster, J. , Davies, D.M. , Julien, S. , Cooper, L. et al. ( 2008 ) Retargeting of human T cells to tumour-associated MUC1: the evolution of a chimeric antigen receptor . J. Immunol. 180 , 4901 – 4909 10.4049/jimmunol.180.7.4901 18354214

45 Posey A.D. Jr , Schwab, R.D. , Boesteanu, A.C. , Steentoft, C. , Mandel, U. , Engels, B. et al. ( 2016 ) Engineered CAR T cells targeting the cancer-associated Tn-Glycoform of the membrane mucin MUC1 control adenocarcinoma . Immunity 44 , 1444 – 1454 10.1016/j.immuni.2016.05.014 27332733 PMC5358667

46 Sørensen A.L. , Reis C.A. , Tarp M.A. , Mandel U. , Ramachandran K. , Sankaranarayan V. et al. ( 2006 ) Chemoenzymatically synthesized multimeric Tn/STn-MUC1 glycopeptides elicit cancer specific anti-MUC1 antibody responses and override tolerance . Glycobiology 16 , 96 – 107 10.1093/glycob/cwj044 16207894

47 Tarp M.A. , Sørensen A.L. , Mandel U. , Paulsen H. , Burchell J. , Taylor-Papadimitriou J. et al. ( 2007 ) Identification of a novel cancer-specific immunodominant glycopeptide epitope in the MUC1 tandem repeat . Glycobiology 17 , 197 – 209 10.1093/glycob/cwl061 17050588

48 Correa I. , Plunkett T. , Vlad A. , Mungul A. , Candelora-Kettel J. , Burchell J.M. et al. ( 2003 ) Form and pattern of MUC1 expression on T cells activated in vivo or in vitro suggests a function in T cell migration . Immunology 108 , 32 – 41 10.1046/j.1365-2567.2003.01562.x 12519300 PMC1782871

49 Gavrill, A. ( 2017 ) Chimeric Antigen Receptor (CAR) T-cell Immunotherapy for MUC1-positive Breast Cancer PhD Thesis , Kings College London

50 Vlad, A.M. , Muller, S. , Cudic, M. , Paulsen, H. , Otvos L. Jr , Hanisch, F.G. et al. ( 2002 ) 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 , 1435 – 1446 10.1084/jem.20020493 12461079 PMC2194269

51 Ryan, S.O. , Vlad, A.M. , Islam, K. , Gariépy, J. and Finn, O.J. ( 2009 ) 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 , 611 – 618 10.1515/BC.2009.070 19426130 PMC4729297

52 Scheid, E. , Major, P. , Bergeron, A. , Finn, O.J. , Salter, R.D. , Eady, R. et al. ( 2016 ) Tn-MUC1 DC vaccination of rhesus macaques and a phase I/II trial in patients with nonmetastatic castrate-resistant prostate cancer . Cancer Immunol. Res. 4 , 881 – 892 10.1158/2326-6066.CIR-15-0189 27604597 PMC5331878

53 Beatson, R. , Tajadura-Ortega, V. , Achkova, D. , Picco, G. , Tsourouktsoglou, T.D. , Klausing, S. et al. ( 2016 ) The mucin MUC1 modulates the tumor immunological microenvironment through engagement of the lectin Siglec-9 . Nat. Immunol. 17 , 1273 – 1281 10.1038/ni.3552 27595232 PMC5257269

54 Pyzer, A.R. , Stroopinsky, D. , Rajabi, H. , Washington, A. , Tagde, A. , Coll, M. et al. ( 2017 ) MUC1-mediated induction of myeloid-derived suppressor cells in patients with acute myeloid leukemia . Blood 129 , 1791 – 1801 10.1182/blood-2016-07-730614 28126925 PMC5813734

55 Rajabi, H. and Kufe, D. ( 2017 ) MUC1-C oncoprotein integrates a program of EMT, epigenetic reprogramming and immune evasion in human carcinomas . Biochim. Biophys. Acta 1868 , 117 – 122 10.1016/j.bbcan.2017.03.003 28302417 PMC5548633

56 Tagde, A. , Rajabi, H. , Bouillez, A. , Alam, M. , Gali, R. , Bailey, S. et al. ( 2016 ) MUC1-C drives MYC in multiple myeloma . Blood 127 , 2587 – 2597 10.1182/blood-2015-07-659151 26907633 PMC4882805

57 Tagde, A. , Markert, T. , Rajabi, H. , Hiraki, M. , Alam, M. , Bouillez, A. et al. ( 2017 ) Targeting MUC1-C suppresses polycomb repressive complex 1 in multiple myeloma . Oncotarget 8 , 69237 – 69249 10.18632/oncotarget.20144 29050200 PMC5642475

58 Bouillez, A. , Rajabi, H. , Jin, C. , Samur, M. , Tagde, A. , Alam, M. et al. ( 2017 ) MUC1-C integrates PD-L1 induction with repression of immune effectors in non-small-cell lung cancer . Oncogene 36 , 4037 – 4046 10.1038/onc.2017.47 28288138 PMC5509481

59 Bouillez, A. , Adeegbe, D. , Jin, C. , Hu, X. , Tagde, A. , Alam, M. et al. ( 2017 ) MUC1-C promotes the suppressive immune microenvironment in non-small cell lung cancer . Oncoimmunology 6 , e1338998 10.1080/2162402X.2017.1338998 eCollection 2017 28932637 PMC5599083

60 Pyzer, A.R. , Stroopinsky, D. , Rosenblatt, J. , Anastasiadou, E. , Rajabi, H. , Washington, A. et al. ( 2017 ) MUC1 inhibition leads to decrease in PD-L1 levels via upregulation of miRNAs . Leukemia 31 , 2780 – 2790 10.1038/leu.2017.163 28555079 PMC5791150

61 Maeda, T. , Hiraki, M. , Jin, C. , Rajabi, H. , Tagde, A. , Alam, M. et al. ( 2018 ) MUC1-C induces PD-L1 and immune evasion in triple-negative breast cancer . Cancer Res. 78 , 205 – 215 10.1158/0008-5472.CAN-17-1636 29263152 PMC5754244

62 Miles, D. , Roché, H. , Martin, M. , Perren, T.J. , Cameron, D.A. , Glaspy, J. et al. ; Theratope® Study Group ( 2011 ) Phase III multicenter clinical trial of the sialyl-TN (STn)-keyhole limpet hemocyanin (KLH) vaccine for metastatic breast cancer . Oncologist 16 , 1092 – 1100 10.1634/theoncologist.2010-0307 21572124 PMC3228158

63 Verheijen, R.H. , Massuger, L.F. , Benigno, B.B. , Epenetos, A.A. , Lopes, A. , Soper, J.T. et al. ( 2006 ) Phase III trial of intraperitoneal therapy with yttrium-90-labeled HMFG1 murine monoclonal antibody in patients with epithelial ovarian cancer after a surgically defined complete remission . J. Clin. Oncol. 24 , 571 – 578 10.1200/JCO.2005.02.5973 16446329