Steffen Goletz

PankoMab: a potent new generation anti-tumour MUC1 antibody

Recently, we described a new carbohydrate-induced conformational tumour-epitope on mucin-1 (MUC1) with the potential for improvement of immunotherapies [29, 30]. PankoMab is a novel antibody, which binds specifically to this epitope and was designed to show the highest glycosylation dependency and the strongest additive binding effect when compared to other MUC1 antibodies. This enables PankoMab to differentiate between tumour MUC1 and non-tumour MUC1 epitopes. It has a high-affinity towards tumour cells (e.g. KD [M] of 0.9 and 3×10−9 towards NM-D4 and ZR75-1, respectively) and detects a very large number of binding sites (e.g. 1.0 and 2.4×106 for NM-D4 and ZR75-1, respectively). PankoMab is rapidly internalised, and after toxin coupling is able to induce very effectively toxin-mediated antigen-specific tumour cell killing. PankoMab reveals a potent tumour-specific antibody-dependent cell cytotoxicity (ADCC). PankoMab is, therefore, distinguished by a combination of advantages compared to other MUC1 antibodies in clinical development, including higher tumour specificity, higher affinity, a higher number of binding sites, largely reduced binding to shed MUC1 from colon and pancreatic carcinoma patients, no binding to mononucleated cells from peripheral blood (except ~7% of activated T cells), stronger ADCC activity and rapid internalisation as required for toxin-mediated cell killing. This renders it a superior antibody for in vivo diagnostics and various immunotherapeutic approaches.

In vitro priming of tumor-specific cytotoxic T lymphocytes using allogeneic dendritic cells derived from the human MUTZ-3 cell line

The adoptive transfer of in vitro-induced and expanded tumor-specific cytotoxic T lymphocytes (CTL) presents a promising immunotherapeutic approach for the treatment of cancer. The in vitro induction of tumor-reactive CTL requires repeated stimulation of CTL precursors with dendritic cells (DC). To circumvent problems like scarcity of blood DC precursors and donor variability, it would be attractive to use DC from a non-autologous, unlimited source. DCs derived from the human acute myeloid leukemia (AML) cell line MUTZ-3 are attractive candidates since these DCs closely resemble monocyte-derived DC (MoDC) in terms of phenotype and T cell stimulatory capacity. Here we demonstrate that functional CTL clones could be generated against multiple tumor-associated antigens, i.e., human telomerase reverse transcriptase (hTERT), ErbB3-binding protein-1 (Ebp1), carcinoembryonic antigen (CEA) and Her-2/neu, by stimulating CD8β+ CTL precursors with peptide-loaded allogeneic, HLA-A2-matched MUTZ-3-derived DC. A consistent induction capacity, as determined by MHC tetramer-binding, was found in multiple donors and comparable to autologous peptide-loaded MoDC. Functional characterization at the clonal level revealed the priming of CTL that recognized endogenously processed epitopes on tumor cell lines in an HLA-A2-restricted fashion. Our data indicate that MUTZ-3-derived DC can be used as stimulator cells for in vitro priming and expansion of functional TAA-specific effector CTL. MUTZ-3-derived DCs thus represent a ready and standardized source of allogeneic DC to generate CTL for therapeutic adoptive transfer strategies.

What Makes MUC1 a Tumor Antigen

The epithelial mucin 1 (MUC1) is an accepted serum tumor marker and cellular tumor antigen. We discuss recent views on the difference(s) between normal and tumor MUC1, and its implication for the development of cancer vaccines and antibody therapies, with special emphasis on the role of glycosylation.

What makes cancer stem cell markers different

Since the cancer stem cell concept has been widely accepted, several strategies have been proposed to attack cancer stem cells (CSC). Accordingly, stem cell markers are now preferred therapeutic targets. However, the problem of tumor specificity has not disappeared but shifted to another question: how can cancer stem cells be distinguished from normal stem cells, or more specifically, how do CSC markers differ from normal stem cell markers? A hypothesis is proposed which might help to solve this problem in at least a subgroup of stem cell markers. Glycosylation may provide the key.

Expression of CD175 (Tn), CD175s (sialosyl-Tn) and CD176 (Thomsen-Friedenreich antigen) on malignant human hematopoietic cells

The expression of the histo‐blood group carbohydrate structures T‐nouvelle (Tn, CD175), sialylated Tn (CD175s) and the Thomsen‐Friedenreich disaccharide (TF, CD176) on human leukemia cell lines was analyzed by their reactivity with specific monoclonal antibodies in flow cytometry, immunohistology and immunoprecipitation. Expression of sialylated CD176 was evaluated by comparative immunostaining with anti‐CD176 antibodies before and after sialidase treatment. While only few cell lines expressed unmasked CD176, sialylated CD176 was present on all hematopoietic cell lines and native lymphocytes examined. CD175 and CD175s are preferentially expressed on erythroblastic leukemia cell lines. CD175s expression in these cells is consistent with the transcription of the gene encoding the key enzyme α2,6‐sialyltransferase (hST6GalNAc1). The staining intensity was reduced after methanol pretreatment of cells, indicating that these glycans are partially expressed as constituents of glycosphingolipids. Immunoprecipitation and subsequent Western blotting revealed a series of distinct high molecular glycoproteins as carriers for these carbohydrate antigens. CD34 was identified as major carrier of CD176 by immunoprecipitation and microsequencing on a KG‐1 subline enriched for CD176 expression. Incubation of several CD176‐positive cell lines with anti‐CD176 antibodies induced apoptosis of these cells, an effect not observed with anti‐CD175/CD175s antibodies. Since the presence of naturally occurring anti‐CD176 antibodies may represent a mechanism of immunosurveillance against CD176‐positive tumor cells, we propose that sialylation of surface‐expressed CD176—among other functions—protects against apoptosis.

Thomsen-Friedenreich Antigen: The “Hidden” Tumor Antigen

Carbohydrate tumor antigens on glycoproteins and glycolipids are targets for active and passive cancer immunotherapy. These highly abundant antigens are de novo expressed or up-regulated due to changes in the complex glycosylation apparatus of tumor cells, involving sets of enzymes like glycosyltransferases, glycosidases, epimerases, and nucleotide sugar transporters. Various lipid or protein bound carbohydrate tumor antigens are described, for example, GM2, GD2, GD3, fucosylated GM1, Globo H, LeY, Lea, Sialyl-Lea and the mucin core structures Tn, Sialyl-Tn, and the Thomsen-Friedenreich Antigen (TF). Carbohydrate tumor antigens are far more abundant than protein tumor antigens rendering them suitable targets especially for antibodies, for example, highly expressed protein tumor markers as Her-2/neu express about 106 and TF about 107 copies per cell. More recent data show that certain carbohydrate structures are not only targets for humoral but also cellular immune responses.

Functional improvement of antibody fragments using a novel phage coat protein III fusion system.

Functional expressions of proteins often depend on the presence of host specific factors. Frequently recombinant expression strategies of proteins in foreign hosts, such as bacteria, have been associated with poor yields or significant loss of functionality. Improvements in the performance of heterologous expression systems will benefit present-day quests in structural and functional genomics where high amounts of active protein are required. One example, which has been the subject of considerable interest, is recombinant antibodies or fragments thereof as expressions of these in bacteria constitute an easy and inexpensive method compared to hybridoma cultures. Such approaches have, however, often suffered from low yields and poor functionality. A general method is described here which enables expressions of functional antibody fragments when fused to the amino-terminal domain(s) of the filamentous phage coat protein III. Furthermore, it will be shown that the observed effect is neither due to improved stability nor increased avidity.

Expression of CD176 (Thomsen-Friedenreich antigen) on lung, breast and liver cancer-initiating cells

The cancer‐initiating capacity of most malignant tumours is considered to reside in a small subpopulation of cells. Therapeutical interventions should target these cells rather than the tumour mass. Numerous studies have shown that the carbohydrate antigen structure CD176 (Thomsen‐Friedenreich antigen, core‐1) is present in many types of cancer and absent in normal adult human tissues. In this study, we assessed whether CD176 is co‐expressed with CD44 or CD133 [markers of cancer‐initiating cells (CIC)] in human lung, breast and liver carcinoma. A variety of human cancer cell lines and surgical specimens of these malignancies were examined. It was found that in most cases the majority of tumour cells stained strongly for CD44 by immunohistochemistry and flow cytometry, whereas CD133 expression was found on a smaller, but varying proportion of cells. Co‐expression of CD176 with CD44 was found at a surprisingly high percentage of cancer cells in vitro and in vivo. Co‐expression of CD176 with CD133 was also detected, although at a lower rate. Tamoxifen treatment of MDA‐435 breast cancer cells enhanced the CD44+/CD176+ phenotype. Evidence is provided through a new sandwich solid‐phase enzyme‐linked immunosorbent assay (ELISA) suggesting that CD44 is a carrier molecule for CD176 not only in colorectal cancer as previously reported, but also in lung, breast and liver cancer. The expression of CD176 in CIC suggests that it may represent an effective target for tumour therapies.

Reactivity of a humanized antibody (hPankoMab) towards a tumor-related MUC1 epitope (TA-MUC1) with various human carcinomas

Humanized antibodies against tumor-related antigens are now established reagents for in vivo diagnostics and for therapeutic approaches, and are increasingly developed. Humanized PankoMab (hPankoMab), a humanized form of PankoMab directed against a novel tumor-related MUC1 epitope (TA-MUC1), was recently developed for clinical application. In the present study, the reactivity of hPankoMab with various human cancers was systematically studied by immunohistochemistry on 137 surgical specimens, including lung, breast, gastric, colorectal, liver, cervical, kidney, thyroid, and other carcinomas, as well as on several non-epithelial malignancies. The study was performed on paraffin sections without antigen retrieval. hPankoMab reacted strongly with carcinomas originating from glandular or squamous epithelia, weakly with hepatocellular carcinomas, and not with sarcomas. The binding sites of hPankoMab in carcinomas were found around the whole cell surface and, in most cases, also in the cytoplasm of cancer cells.

Co-expression of CD173 (H2) and CD174 (Lewis Y) with CD44 suggests that fucosylated histo-blood group antigens are markers of breast cancer-initiating cells

Histo-blood group antigens CD173 (H2) and CD174 (Lewis Y) are known to be developmentally regulated carbohydrate antigens which are expressed to a varying degree on many human carcinomas. We hypothesized that they might represent markers of cancer-initiating cells (or cancer stem cells, CSC). In order to test this hypothesis, we examined the co-expression of CD173 and CD174 with stem cell markers CD44 and CD133 by flow cytometry analysis, immunocytochemistry, and immunohistochemistry on cell lines and tissue sections from breast cancer. In three breast cancer cell lines, the percentage of CD173+/CD44+ cells ranged from 17% to >60% and of CD174+/CD44+ from 21% to 57%. In breast cancer tissue sections from 15 patients, up to 50% of tumor cells simultaneously expressed CD173, CD174, and CD44 antigens. Co-expression of CD173 and CD174 with CD133 was also observed, but to a lesser percentage. Co-immunoprecipitation and sandwich ELISA experiments on breast cancer cell lines suggested that CD173 and CD174 are carried on the CD44 molecule. The results show that in these tissues CD173 (H2) and CD174 (LeY) are associated with CD44 expression, suggesting that these carbohydrate antigens are markers of cancer-initiating cells or of early progenitors of breast carcinomas.