Table 1 Targets expressed on tumour initiating cells

EGFRvIII

Albert Wong, Stanford University Medical Center

• Truncated version of EGFR, with autonomous signalling and pro-tumorigenic capabilities

• Expressed by CSCs in glioblastoma: the cells with the highest tumorigenic properties are CD133 EGFRvIII double-positive cells

• Neither homogenously expressed within tumors, nor indispensable to tumor cells; its expression could vary during tumor progression

• EGFRvIII is being pursued through vaccination, monoclonal antibodies and adoptive T cell therapy with CARs

Cyclin A1

Wilms Tumor antigen -1 (WT-1)

Philip Greenberg, University of Washington, Fred Hutchinson Cancer Research Center

• Cyclin A1 and WT-1 are expressed in Acute Myeloid Leukemia (AML) CSCs

• Cyclin A1 is a new target: its expression encoded by CCNA1 is largely restricted to the meiotic phase in normal germinal cells but appears to be co-opted by many malignancies, including ~60% of cases of AML

• T cells against Cyclin A1 and WT-1 epitopes were generated and tested in preclinical models

• Clinical evaluation of TCR-engineered adoptive T cell therapy is ongoing in AML patients with antigen⁺ leukemia and the appropriate HLA restricting element

Chondroitin sulfate proteoglycan 4 (CSPG4)

Soldano Ferrone, Massachusetts General Hospital and Harvard Medical School

• Complex and extensively glycosylated tumor antigen expressed on the cell membrane

• Amenable to immune interventions such as antibody therapy and chimeric antigen receptor (CAR)-engineered T cells

• Expressed on normal cells and highly upregulated on tumor cells of various origin: ectodermic, endodermic and mesodermic

• Within tumors, CSPG4 could be also expressed on pericytes and other stromal cells, supporting a multi-pronged mechanism of action

• CSPG4 is also expressed on tumor initiating cells

• Its expression on some normal cells associated with vasculature and central nervous system could be of concern; yet antibody approaches directed to post-translational modifications could be a fertile area of new drug development

5T4 -trophoblast glycoprotein (TPGB)

Kenneth Geles, Pfizer Inc

• Tumor-specific membrane expression, amenable to antibody therapy. Normal expression of is limited to placenta and embryonic stem cells

• 5T4 is over-expressed in colorectal, gastric and ovarian cancers and is associated with advanced disease and/or worse clinical outcome

• It can function as a pro-migratory factor in embryonic cells that have undergone an epithelial-to-mesenchymal (EMT) transition and can also modulate CXCR4 and Wnt signalling

• 5T4 is also enriched on cancer stem cells (tumor-initiating cells) in non-small cell lung carcinoma (NSCLC)

• In the H460 lung cancer cell line, the CD24^low/CD44^high subset is most tumorigenic and enriched for the 5T4 mRNA

• Sorting cells from a NSCLC patient derived xenograft (PDX) based on 5T4 expression confirmed that 5T4^high cells were more tumorigenic

• High levels of 5T4 expression were associated with poorly differentiated NSCLC tumors and worse overall survival

• Treatment of preclinical lung and breast cancer models with an anti-5T4 antibody drug conjugate (A1-mcMMAF) resulted in long-term tumor regressions. This is the first proof-of-concept targeting a heterogeneous subpopulation of cells at the apex of a cellular hierarchy with an ADC

• This therapeutic candidate entered Phase 1 clinical trials

Brachyury

Claudia Palena, National Cancer Institute

• Brachyury is an embryonically relevant T-box protein required for the normal development of the mesoderm

• It is aberrantly expressed in various tumor types, including lung, breast, colon and prostate carcinomas

• Primary tumors, metastatic lymph nodes

• and distant metastasis of breast cancer have been shown to be highly positive for Brachyury

• Expression of Brachyury in epithelial cancer cells drives EMT. Tumor cells undergoing EMT acquire features of stemness

• Expression of Brachyury in lung and

• breast carcinoma cells has been associated with resistance to conventional anti-cancer modalities

• As antibodies are not applicable and small molecules have been unsuccessful, T-cell mediated immunotherapeutic approaches against Brachyury are being developed

• Brachyury has been shown to be an immunogenic molecule; an HLA-A0201 epitope was identified (WLLPGTSTL) and used to efficiently expand Brachyury-specific CD8+ T cells from patients

• Brachyury-based cancer vaccine is in clinical development

HER-2/Neu

Brian Czerniecki, University of Pennsylvania

• Well characterized cell surface, cell growth receptor within the EGFR class of receptors, with amplified expression and prominent biology

• Expressed on CSC in luminal breast cancer

• When co-expressed with the estrogen receptor, HER-2 expression is up-regulated by mechanisms other than gene amplification

• CSC with dim Her-2 expression could have a role in tumor escape from targeted therapies, irrespectively of the Her-2 status of the primary tumor

• Her-2-directed antibody therapies are not applicable or not effective in patients with tumors that have modest levels of Her-2 expression

• Thus, alternate immune interventions to target such cells are needed, such as therapeutic vaccines, currently in development

EZH2

Elaine Hurt, Medimmune

• Described an in vitro high throughput assay to discover novel targets associated with cancer stem cells

• These targets are amenable to antibody based immunotherapy and related approaches, as they are expressed on the cell membrane

• Described EZH2, a novel target associated with the Wnt/Notch pathway, and thus closely related to stemness

Cancer testes antigens (CTAs)

John Yu, Immunocellular Therapeutics

• Another source of cancer stem cell-associated targets is represented by glioblastoma, with convincing evidence in support of their existence

• These cells are largely chemorefractory and radioresistant, and responsible for tumor relapse

• As this cancer originates from the ectoderm, its shares antigens with melanoma. CTAs with restricted expression within normal vital organs, constitute a rich source of novel targets amenable to immunotherapy

• An approach has been designed to target such antigens by utilizing active immunotherapy (therapeutic cancer vaccination) to elicit multiple immune responses against glioblastoma cancer stem cells

Maurizio Chiriva-Internati, Texas Tech University

• Most CTAs characterized to date are expressed inside the cell and thus are targetable only through immunotherapies that are directed at MHC-restricted epitopes (therapeutic vaccines and TCR-engineered T cells)

• There is emerging evidence that certain CTAs, associated with tumor initiating cells, can be also displayed onto the cell membrane

• Such targets are SP17, AKAP4, Ropporin, and PTTG1, expressed in a broad range of tumors of widely different histological origin: multiple myeloma, lung cancer, ovarian and prostate carcinoma

• AKAP4 in particular showed some promising evidence of membrane expression in multiple myeloma cells

• The possible expression of some CTAs onto the cell membrane, render these molecules promising targets for antibody and CAR-approaches