Ludwig Institute for Cancer Research

Publications

LICR Investigators Pivotal in Five of the

24 Milestones in Cancer Research

In 2006, the Nature Publishing Group gave its opinion on the ‘24 Milestones in Cancer Research’ since the end of the nineteenth century. ‘Original Research Papers’ authored by LICR investigators feature in five of the 24 Milestones, with another two quoting papers authored by LICR investigators as selected ‘Further Reading.’ That’s about 30% of cancer research milestones with significant contributions from LICR investigators according to the editors of Nature, Nature Medicine and Nature Reviews Cancer.

References

1. Van Pel A. and Boon T. Protection against a nonimmunogenic mouse leukemia by an immunogenic variant obtained by mutagenesis. Proc Natl.Acad.Sci.U.S.A (1982) 79(15):4718-4722.


2. van der Bruggen P., Traversari C., Chomez P., Lurquin C., De Plaen E., Van den E.B., Knuth A., and Boon T. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science (1991) 254(5038):1643-1647.


3. Shankaran V., Ikeda H., Bruce A.T., White J.M., Swanson P.E., Old L.J., and Schreiber R.D. IFNgamma and lymphocytes prevent primary tumour development and shape tumour immunogenicity. Nature (2001) 410(6832):1107-1111.[PMID: ]


4. Cavenee W.K., Dryja T.P., Phillips R.A., Benedict W.F., Godbout R., Gallie B.L., Murphree A.L., Strong L.C., and White R.L. Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature (1983) 305(5937):779-784.


5. Waterfield M.D., Scrace G.T., Whittle N., Stroobant P., Johnsson A., Wasteson A., Westermark B., Heldin C.H., Huang J.S., and Deuel T.F. Platelet-derived growth factor is structurally related to the putative transforming protein p28sis of simian sarcoma virus. Nature (1983) 304(5921):35-39


6. Downward J., Yarden Y., Mayes E., Scrace G., Totty N., Stockwell P., Ullrich A., Schlessinger J., and Waterfield M.D. Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature (1984) 307(5951):521-527.


7. DeLeo A.B., Jay G., Appella E., DuBois G.C., Law L.W., and Old L.J. Detection of a transformation-related antigen in chemically induced sarcomas and other transformed cells of the mouse. Proc.Natl.Acad.Sci.U.S.A (1979) 76(5):2420-2424.


8. Gessler M., Poustka A., Cavenee W., Neve R.L., Orkin S.H., and Bruns G.A. Homozygous deletion in Wilms tumours of a zinc-finger gene identified by chromosome jumping. Nature (1990) 343(6260):774-778.


9. Bronner C.E., Baker S.M., Morrison P.T., Warren G., Smith L.G., Lescoe M.K., Kane M., Earabino C., Lipford J., Lindblom A., Tannergard P., Bollag R.J., Godwin A.R., Nordenskjosolld M., Fishel R., Kolodner R., and Liskay R.M. Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer. Nature (1994) 368(6468):258-261.


10. Fishel R., Lescoe M.K., Rao M.R., Copeland N.G., Jenkins N.A., Garber J., Kane M., and Kolodner R. The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell (1993) 75(5):1027-1038.


11. Demetri G.D., von Mehren M., Blanke C.D., Van den Abbeele A.D., Eisenberg B., Roberts P.J., Heinrich M.C., Tuveson D.A., Singer S., Janicek M., Fletcher J.A., Silverman S.G., Silberman S.L., Capdeville R., Kiese B., Peng B., Dimitrijevic S., Druker B.J., Corless C., Fletcher C.D., and Joensuu H. Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N.Engl.J.Med. (2002) 347(7):472-480.

From discovery of proteins to multiple potential therapies

Angiogenesis and VEGFs

Angiogenesis, the process of forming new blood vessels, is central to wound healing, reproduction and embryonic development. Tumor cells and stroma, the connective tissue around the tumor, can also stimulate angiogenesis by secreting angiogenic growth factors. Without the blood and nutrients supplied by newly-generated blood vessels, it is thought that no tumor would grow to be more than a few millimeters in size. Lymphangiogenesis, a related process, is the formation of new lymphatic vessels, which is also stimulated by particular growth factors. The blood and lymphatic vessels provide the principle routes by which cancer metastasizes from the original tumor site, which is the ultimate cause of most cancer deaths.

For more than ten years, LICR coordinated and supported a global Program to study angiogenesis and lymphangiogenesis with the aim of developing new therapeutic modalities. LICR efforts identified three of the four known vascular endothelial growth factors (VEGFs), VEGF-B, VEGF-C and VEGF-D, two of the four known platelet-derived growth factors (PDGFs), PDGF-C and PDGF-D, as well as a novel receptor, VEGF receptor-3 (VEGFR-3).

LICR conducted extensive laboratory and pre-clinical studies to assess the biological functions of the growth facotors and the therapeutic potential related to the discovery of the VEGFs and PDGFs. For example, LICR investigators were the first to show that antibodies targeting VEGF ligands inhibit cancer spread to the lymph nodes, suggesting that anti-VEGF antibodies are potential anti-metastatic therapies. Vegenics Ltd (Australia), an LICR spin-off company that is now a wholly-owned subsidiary of Circadian Technologies Ltd (Australia), is developing antagonists of VEGF-C and VEGF-D as both therapeutic and diagnostic agents for cancer.

LICR has a singular focus on cancer but recognizes that some of its medical research findings may have therapeutic value for other human diseases. Thus LICR places great importance on supporting and facilitating—principally through the licensing of its intellectual property—the research and development of non-oncology therapies for human benefit. Ark Therapeutics PLC is developing a pro-angiogenic therapy, Trinam®, which utilizes VEGF-D licensed from LICR.  Trinam® is currently being tested in a phase III clinical trial for its ability to prevent blood vessel blockage following vascular graft access surgery (insertion of an artificial blood vessel), which is required for patients with kidney failure to undergo dialysis. Lymphatix (Finland), the second company spun-off from the Angiogenesis Program, was recently purchased by Ark Therapeutics.

Study of natural history of infection to preventative vaccine

HPV Cervical Cancer Vaccine

With its flexible, international reach, LICR was able to access a large number of cancer samples in order to analyze the natural history or “epidemiology” of infection with human papillomavirus (HPV), the causative agent of genital cancers, with a view to preventing and/or treating these diseases.

LICR began studying the role of HPV in cancers of the cervix, penis and anus in the early 1980’s and found that DNA sequences from HPV were very often found in these genital tumors. This finding, along with others from around the world contributed to the confirmation that HPV is the causative agent of cervical cancer. Dr. Harald zur Hausen—a former member of the LICR Scientific Advisory Committee—was awarded the 2008 Nobel Prize for Medicine & Physiology for first postulating and then proving this theory.

However, the discovery of a high proportion of HPV infections in asymptomatic women led LICR to launch epidemiological studies aimed at understanding the risk correlates for HPV-associated cervical disease.

In 1993, the “Ludwig/McGill Cohort” was established by LICR investigators with a population of women from São Paulo, Brazil. This cohort formed one of the largest longitudinal studies of the natural history of HPV infection and risk of cervical cancer in the world. (The McGill part of the name pays tribute to the co-director of the cohort epidemiologist who worked at the LICR São Paulo Branch before moving to McGill University in Canada.)

LICR investigators found that most HPV infections are transient and of little clinical significance. However, the small proportion of women who harbor persistent HPV infections stand at a much greater risk of subsequent cervical neoplasia, indicating that persistent, not transient, HPV infections are the actual biologic precursor in cervical cancer onset. This and other findings on viral load (the amount of virus) and the integration of viral DNA into cervical cells were important for understanding the clinical significance of HPV DNA testing results and set policies for inclusion of some form of HPV testing in cervical cancer prevention internationally.

Results from LICR’s HPV natural history studies were seminal in facilitating the design and implementation of clinical trials of prophylactic vaccines against HPV. The LICR team was invited to lead a phase II trial of Merck & Co. Inc.’s prophylactic (preventative) HPV vaccine, which was the first study to demonstrate the safety, immunogenicity and efficacy of the vaccine. The vaccine—Gardasil®—was approved for licensing later that year (2005) and is now sold internationally.

From brain cancer initiative to new category of antibody target

EGFR and Antibody 806

Several antibodies that target the cell surface signaling molecules promoting cancer cell growth have been approved for cancer patients. However, many of the first generation antibodies currently in clinical use do not discriminate between the signaling molecules on cancer cells and those on healthy cells. This lack of discrimination is the apparent cause of the toxic side-effects associated with these drugs.

The epidermal growth factor receptor (EGFR) is a prime target for the development of anti-cancer therapeutics, as EGFR overexpression (increased abundance) or mutation has been reported in numerous human cancers and is generally associated with a poor clinical prognosis. Anti-EGFR antibody therapies are available, but they cause skin and liver toxicities. Most cases of glioblastoma (brain cancer) are caused by a particular EGFR mutation, and LICR investigators set out to generate an antibody that specifically targeted the mutated EGFR in the hope of developing an effective therapy for a disease that is intractable to all conventional treatments.

LICR investigators found that the antibody, 806, does indeed target the mutation found in glioblastoma. However, extensive laboratory and pre-clinical analyses showed that 806 is also able to discriminate between wild-type (non-mutated) EGFR and over-expressed wild-type EGFR. Further characterization showed that the 806 antibody is able to differentiate between cell surface proteins with identical sequences but different conformations (shapes) resulting from the over-expression of the receptor.

By taking advantage of the unique binding specificity of the 806 antibody, LICR investigators further unraveled the complexities of EGFR activation and its hyper-activity in cancer. Their discovery that EGFR exposes the binding site for 806 as it changes from an inactive form to an active form was a major advance in the understanding of growth receptor activation. These surprising but pivotal findings identified a whole new category of cell surface target for antibody-based cancer therapies.

LICR sponsored and conducted the first-in-man clinical trial to characterize 806’s tumor-targeting abilities and showed that it targets the tumors but not normal tissues that have high levels of EGFR. The clinical research also showed—presumably as a result of its specificity—806 does not cause the side-effects observed with other EGFR-targeting therapies, e.g. Erbitux (Cetuximab).

The 806 antibody formed the basis of an LICR spin-off company, Life Sciences Pharmaceuticals, and in 2009 was licensed to the pharmaceutical company Abbott for clinical development.

Cancer Vaccine Collaborative

The Ludwig Institute for Cancer Research Ltd (LICR) and the Cancer Research Institute (CRI) (New York, USA) joined forces in 2001 to form the Cancer Vaccine Collaborative (CVC). Therapeutic cancer vaccines represent an entirely new therapeutic modality with great promise for helping to reduce tumor recurrence. By working together within the CVC, LICR and CRI investigators are able to best achieve their common objectives of understanding the immunological response to cancer, harnessing that knowledge for patient benefit, and accelerating the translation of basic research into new cancer therapies.

The CVC provides an international clinical trial infrastructure (the LICR Office of Clinical Trials Management) to help investigators navigate the complex challenges of translating their laboratory findings into the clinic. These activities include the writing and filing of complex regulatory documentation, obtaining or manufacturing clinic-grade reagents, procuring funding for patient costs, meeting insurance requirements, and fulfilling stringent clinical trial monitoring requirements. The CVC also supports the associated laboratory experiments that enable the investigator to understand the mechanism of the tested therapy; research that goes beyond the primary or secondary endpoints of the typical clinical trial.

Since its inception, the CVC has completed or is currently conducting 35 clinical trials. These trials have enrolled nearly 700 patients with a variety of cancer types, including melanoma and sarcoma, and ovarian, prostate, lung, breast, esophageal, and bladder cancers. With the progression of several of these studies into Phase II and III trials, the CVC has become a leading force in the development of therapeutic cancer vaccines as a real option for cancer patients.

Strategic Translation of Knowledge Yields Potential Therapies

PI3K Discovery to Clinic

The PI3K signaling pathway, which regulates several vital cell and physiological processes, is the most frequently subverted signaling pathway in cancer. LICR’s long-term and substantial commitment to PI3K research demonstrates how laboratory discoveries can be translated into applications for human benefit. The Institute’s investment—in staff, research funds and time, academic and industry relationships, intellectual property and technology licensing, and the careful management of their integration—has thus far brought several candidate therapies to late-stage clinical development for cancer and other disease applications.

There are 14 proteins in the PI3K family, and LICR investigators discovered nearly half of that number. LICR also established the internationally adopted nomenclature for the PI3K enzymes by classifying the various PI3K isoforms based on their structure and function.

Pivotal studies by LICR researchers established the roles of signaling by different PI3K isoforms in: cell processes, such as growth, proliferation, migration, and growth factor receptor signaling; physiological processes, such as inflammation and immunology; and in diseases from solid tumors and leukemia to arthritis, obesity and diabetes.

With PI3K signaling so heavily implicated in cancer onset and progression, PI3K isoforms were considered prime targets for potential new therapies. However, the ubiquity of PI3K signaling in normal cell processes means that a therapeutic modality targeting all PI3K signaling would likely cause severe side-effects. One of the most fundamental contributions to the development of PI3K inhibitors was the LICR”s generation of mouse models in which specific PI3K isoforms had been inactivated. The inactivated PI3K isoforms mimic the effects of systemic administration with a subunit-specific drug, and a careful assessment of these mice provided crucial information for assessing potential side-effects from PI3K inhibition. The finding that mice with an inactive p110alpha PI3K, which is most often implicated in cancer onset and progression, had dampened insulin signaling but no signs of developing diabetes or severe metabolic disturbances, was proof-of-principle that isoform-specific inhibitors were indeed candidate cancer therapies.

LICR’s first spin-off company, PIramed Ltd, was formed to develop PI3K inhbitors generated through a translational research program initiated by LICR. The collaboration—between academic partners LICR, Imperial Cancer Research Fund (now Cancer Research UK) and the Institute for Cancer Research (both in London, UK), and industry partner Yamanouchi Pharmaceutical Company (now Astellas Pharma, Japan)—generated multiple lead compounds that were screened for isoform specificity and also physiological effect. This approach of combining fundamental science with translational work advanced our knowledge of PI3K signaling in cancer and other diseases and also enabled the industrial development team to make informed decisions based on solid science. PIramed entered a major development deal with Genentech before being acquired by Roche in one of the largest deals in the UK biotechnology sector for some time. An isoform-specific inhibitor of PI3K p110alpha is now in a phase I trial as a potential cancer therapy.

Cell Biology

Stem Cells: New Prospects for Cancer Therapy

Stem cells—unspecialized cells with high capacity for self-renewal—are present in both embryonic and adult tissues and can differentiate to acquire distinct shapes and functions. Adult stem cells are maintained throughout life to replenish dying cells or repair damaged tissue. In contrast to embryonic stem cells, which can develop into a broad spectrum of cell types, adult stem cell populations can differentiate into fewer different types of cell. Cells with stem-cell like characteristics are also found in tumors. Many hypothesize that these ‘cancer stem cells’ generate new tumors and drive tumor progression with their ability to divide uncontrollably and transform into different cancer cell types. If this is the case, therapies that target cancer stem cells might represent the most effective strategy for destroying tumors.

Dormant Hematopoietic Stem Cells Awaken to Repair Bone Marrow Injury

Hematopoietic stem cells (HSCs) are adult stem cells that differentiate into a range of blood cell types in the bone marrow. LICR Lausanne Branch investigators were the first to report that a substantial number of HSCs are dormant but become active in response to physiological stress. Studies in mice revealed that dormant HSCs divide as rarely as five times in a lifespan under normal conditions. However, the cells are induced to self-renew rapidly by bone marrow injury or by treatment with G-CSF, a cell-to-cell signaling molecule that was discovered by LICR Melbourne Branch investigators many years ago. When physiological balance has been re-established, the HSCs return to dormancy¹.

The hypothesis that dormant HSCs make up an emergency stem cell reserve that can be activated could have important ramifications for cancer therapy strategy. Cancer stem cells can evade eradication by standard chemotherapies that target rapidly diving cells. If cancer stem cells, like HSCs, can be awakened out of dormancy, they could be sensitized to chemotherapy treatment by stem cell activating molecules.

Wnt Signaling in Hematopoietic Stem Cells

The differentiation of HSCs into different blood cell types—a process known as hematopoiesis—is regulated by a group of cell-to-cell signaling proteins known as Wnts. Binding of Wnt to its receptor stabilizes β-catenin and γ-catenin proteins, which mediate the transient activation of Wnt responsive genes. In cancer and some other diseases, Wnt signaling is deregulated by aberrant stabilization of β-catenin or overexpression of γ-catenin, which leads to the uncontrolled expression of Wnt target genes.

Although drugs that target β- or γ-catenin could potentially block aberrant Wnt signaling in cancer cells, there is a risk that hematopoiesis, which is required throughout adult life, could be impaired. However, more research from the LICR Lausanne Branch suggests that β- and γ-catenin can be targeted with no effect on hematopoiesis. The investigators studied mice that lack β- and γ-catenin, and found that hematopoiesis still occurs normally, presumably because the signals required for hematopoiesis are transmitted by other molecules in HSCs². This finding supports the feasibility of therapeutic strategies that target β- or γ-catenin for leukemia patients.

Maintenance of Neural Stem Cells

Neural stem cells, another type of adult stem cell, differentiate into a range of nerve cell types. Neurogenesis—the differentiation of neural stem cells into neurons—is being studied by LICR investigators seeking to identify the mechanisms that keep the neural stem cells’ delicate balance between self-renewal and differentiation into neurons. Several years ago, investigators at the LICR Stockholm Branch discovered that the maintenance of neural stem cells in the central nervous system depends on a group of transcription factors called SoxB1 proteins, as well as on cell signaling by the cell-surface receptor Notch. (Investigators at the LICR Lausanne Branch are also studying Notch signaling, but in T cell differentiation and maturation³.) Although Notch and SoxB1 have similar effects, the extent of their interactions, if any, were unknown.

In 2008, the Stockholm Branch team found that Notch and SoxB1 operate through distinct mechanisms when maintaining neural cells in an undifferentiated state. However, there is some interplay, as Notch’s role in this function is dependent on the SoxB1 proteins⁴. Drawing on this knowledge, the investigators are now exploring ways to target cancer stem cells in the search for new treatment strategies for brain cancer.

References

1. Wilson A, Laurenti E, Oser G, van der Wath RC, Blanco-Bose W, Jaworski M,Offner S, Dunant CF, Eshkind L, Bockamp E, Lió P, Macdonald HR, Trumpp A. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell (2008) 135(6):1118-29 [PMID: 19062086].
2. Jeannet G, Scheller M, Scarpellino L, Duboux S, Gardiol N, Back J, Kuttler F, Malanchi I, Birchmeier W, Leutz A, Huelsken J, Held W. Long-term, multilineage hematopoiesis occurs in the combined absence of beta-catenin and gamma-catenin. Blood (2008) 111(1):142-9 [PMID: 17906078].
3. Fiorini E, Ferrero I, Merck E, Favre S, Pierres M, Luther SA, MacDonald HR. Cutting edge: thymic crosstalk regulates delta-like 4 expression on cortical epithelial cells. J Immunol (2008) 181(12):8199-203 [PMID: 19050235].
4. Holmberg J, Hansson E, Malewicz M, Sandberg M, Perlmann T, Lendahl U, Muhr J. SoxB1 transcription factors and Notch signaling use distinct mechanisms to regulate proneural gene function and neural progenitor differentiation. Development (2008) 135(10):1843-51 [PMID: 18417619].

Cancer Immunology

Immune Responses to Cancer

In addition to protecting the body against infection, the immune system has the capacity to defend against cancer. White blood cells known as cytotoxic T lymphocytes (CTLs) can distinguish between cancer and normal cells by virtue of cancer antigens, specific peptides (protein fragments) presented on the cell’s surface by major histocompatibility complex (MHC) molecules. The CTL’s T cell receptors (TCRs) recognize cells presenting cancer antigen-derived peptides and induce the immunological destruction of the cells.

Therapeutic cancer vaccines are being designed to generate and strengthen CTL responses against cells presenting cancer antigens. In order to optimize cancer vaccines currently in clinical trials and to develop new vaccine strategies, LICR investigators are examining how immune responses to cancer are established and maintained, and also how they can be subverted by tumors.

Immune Suppression by Regulatory T Cells

In spite of the immune system’s inherent capacity to defend against cancer, tumors can develop means to evade immune recognition or escape immunological attack. A type of white blood cell known as a regulatory T cell (Tregs) has been shown to suppress the activity of CTLs in several tumors. Tregs have an important role in preventing auto-immunity¹, the immune system’s attack on normal cells, but they have also been shown to stifle the immunological response against tumors.

The protein FoxP3 plays a critical role in the development and function of Tregs. LICR Lausanne Branch researchers found new evidence that tumors stimulate the formation of Tregs, and that Tregs are attracted to the tumor site(s). The study showed that, compared to healthy donors, melanoma patients have increased amounts of FoxP3-expressing Tregs circulating in their blood and that Tregs were enriched significantly at tumor sites and in tumor-infiltrated lymph nodes².

FoxP3 was believed to be produced exclusively by Tregs, and is used as the most reliable marker to distinguish Tregs from other cell types. However, a study from the LICR Melbourne Center revealed that this protein is also produced by cancer cells. The investigators detected FoxP3 in samples from melanoma and other tumor types, including those from breast, colon, lung, prostate, kidney, and bladder cancers. The team also discovered that melanoma cells produce their own version of FoxP3, which lacks a segment of the protein produced by Tregs and also has an altered sequence³. These findings suggest an unknown mechanism by which tumors might suppress the activity of infiltrating immune cells to escape destruction. They also suggest that a therapeutic approach that combines a cancer vaccine with strategies targeting FoxP3 might be more effective than a vaccine alone.

New Concept of T Cell Receptor Recognition

A mechanism that may modulate CTL activity was uncovered by investigators at the LICR Lausanne Branch. MHC I-peptide complexes displayed on the surface of cells are not fixed in one position but move around considerably within the cell membrane. The TCR briefly engages MHC I-peptide complexes and scans it to determine if the peptide is “non-self,” i.e. derived from a cancer antigen or from a pathogen. Each TCR recognizes one specific MHC I-peptide complex, which is known as its “cognate.” (A separate study from the Lausanne Branch identified, with great precision, the two amino acid residues in one cancer antigen peptide that interact with the TCR⁴.) The LICR researchers studying MHC I-peptide complex mobility found that when the TCR recognizes its cognate complex, the complex becomes immobilized. The trapping of MHC I-peptide complexes is mediated by the adhesion molecule ICAM-1, which is concentrated in the contact sites formed by CTLs and target cells⁵. The temporary capture of the MHC I-peptide complexes allows the TCR to scan the target cells and thus increases the efficiency of TCR antigen recognition. The LICR team is now investigating if defects in the trapping and scanning mechanisms might account for the poor recognition of some tumor cells by CTLs.

Addressing Anergy to Enhance Cancer Vaccines

The ability of tumors to evade immune attack is manifested in part by a phenomenon known as CTL anergy, in which CTLs at the tumor site experience the loss of their ability to recognize cancer cells. In 2008, a LICR Brussels Branch team made pioneering findings that clarify a molecular basis of CTL anergy and how it is achieved by tumors. The study was featured on the front cover of the March 2008 issue of the journal Immunity⁶.

CTLs generally express on their surface a receptor, CD8, that helps the TCR to recognize MHC-peptide complexes on their target cells. In normal CTLs, CD8 receptors are associated with TCRs on the T cell surface. The Brussels Branch team discovered that CD8 and TCR molecules become separated on anergic CTLs, probably because TCR is bound to the protein galectin-3. The investigators were able to restore the association of CD8 and TCR by administering the galectin-binding molecule LacNAc to anergic CTLs. As a result, the CTLs regained their ability to recognize and destroy cancer cells. These findings suggest that treatment of cancer patients with galectin ligands might correct the anergy of tumor-infiltrating lymphocytes. The LICR Brussels Branch team will extend this work into the clinical discovery phase by conducting a clinical trial with a melanoma vaccine that incorporates galectin-binding sugars.

References

1. Jandus C, Bioley G, Rivals JP, Dudler J, Speiser D, Romero P. Increased numbers of circulating polyfunctional Th17 memory cells in patients with seronegative spondylarthritides. Arthritis Rheum (2008) 58(8):2307-17 [PMID: 18668556].
2. Jandus C, Bioley G, Speiser DE, Romero P. Selective accumulation of differentiated FOXP3(+) CD4 (+) T cells in metastatic tumor lesions from melanoma patients compared to peripheral blood. Cancer Immunol Immunother (2008) 57(12):1795-805 [PMID: 18414854].
3. Ebert LM, Tan BS, Browning J, Svobodova S, Russell SE, Kirkpatrick N, Gedye C, Moss D, Ng SP, MacGregor D, Davis ID, Cebon J, Chen W. The regulatory T cell-associated transcription factor FoxP3 is expressed by tumor cells. Cancer Res (2008) 68(8):3001-9 [PMID: 18413770].
4. Derré L, Bruyninx M, Baumgaertner P, Ferber M, Schmid D, Leimgruber A, Zoete V, Romero P, Michielin O, Speiser DE, Rufer N. Distinct sets of alphabeta TCRs confer similar recognition of tumor antigen NY-ESO-1157-165 by interacting with its central Met/Trp residues. Proc Natl Acad Sci USA (2008) 105(39):15010-5 [PMID: 18809922].
5. Segura JM, Guillaume P, Mark S, Dojcinovic D, Johannsen A, Bosshard G, Angelov G, Legler DF, Vogel H, Luescher IF. Increased mobility of MHC I – peptide complexes decreases the sensitivity of antigen recognition. J Biol Chem (2008) 283(35): 24254-63 [PMID: 18579518].
6. Demotte N, Stroobant V, Courtoy PJ, Van Der Smissen P, Colau D, Luescher IF, Hivroz C, Nicaise J, Squifflet JL, Mourad M, Godelaine D, Boon T, van der Bruggen P. Restoring the association of the T cell receptor with CD8 reverses anergy in human tumor-infiltrating lymphocytes. Immunity (2008) 28(3):414-24 [PMID: 18342010].

Cancer Immunology

Identification of New Antigen Targets

Cancer antigens are molecules that are i) present in cancer cells and ii) recognized and targeted by the immune system. Cancer antigens that have limited expression in normal adult tissues are compelling targets for the development of immunotherapies, such as targeted antibodies and therapeutic cancer vaccines (TCVs).

With the discovery of MAGE-A1 in 1991, LICR investigators were the first to identify a unique family of cancer antigens known as the cancer / testis (CT) antigens. The CT antigens—also referred to as cancer/germline antigens—were named according to their selective expression in various cancer and germline (testis and fetal ovary) cells, but not normal adult cells. In the intervening decades, LICR investigators discovered many more CT antigens, characterized their expression, and determined their immunogenicity in both healthy volunteers and cancer patients. LICR has conducted more than 60 clinical trials to explore the therapeutic potential of TCVs based on CT antigens. Laboratory research is ongoing to elucidate the function of these proteins and their possible role(s) in cancer onset and progression.

A Promising New Target for Prostate Cancer Immunotherapy

Prostate cancer detected at an early stage is generally a treatable disease. However, five year survival rates decline sharply to levels near 30% once the cancer metastasizes from the prostate gland. In 2008, research published by a team of investigators at the LICR São Paulo Branch identified the CT antigen CTSP-1 as a candidate for the development of an immunotherapy for prostate cancer¹. The team showed that CTSP-1 protein was expressed in a majority (61%) of the 49 tumor samples analyzed. Additionally, CTSP-1 induced an antibody response in 25% of the 147 prostate cancer patients tested. The LICR team also discovered that detection of anti-CTSP-1 antibodies may be a useful prognostic marker, as the presence of anti-CTSP-1 antibodies was shown to be a powerful indicator of better outcome in patients with aggressive cancers. A TCV against CTSP-1 would hold great promise for the control of metastatic prostate disease, for which new treatments are greatly needed.

Characterization of an Embryo-Cancer Antigen in Non-Small Cell Lung Cancer

In 2008, LICR investigators from the LICR Melbourne Center and New York Branch characterized the expression of the embryo cancer sequence A (ECSA) gene, which is also known as developmental pluripotency associated-2 (DPPA2)². From analysis of more than 300 tumor samples and normal tissues, the team determined that ECSA/DDPA2 expression is limited in normal cells, but occurs in a variety of tumors, most notably non-small cell lung cancer (NSCLC). Additionally, ECSA/DPPA2 protein is co-expressed with several CT antigens in the NSCLC samples and induced a spontaneous anti- ECSA/DPPA2 immune response in some NSCLC patients. Based on the tumor-selective expression of this gene and the knowledge that it is associated with embryogenesis, rather than gametogenesis, the team proposed that ECSA/DPPA2 be classified as an “embryo-cancer antigen” as opposed to a CT antigen. Efforts are underway to evaluate ECSA/DPPA2 as a potential target for NSCLC immunotherapy.

References

1. Parmigiani RB, Bettoni F, Grosso DM, Lopes A, Cunha IW, Soares FA, Carvalho AL, Fonseca F, Camargo AA. Antibodies against the cancer-testis antigen CTSP-1 are frequently found in prostate cancer patients and are an independent prognostic factor for biochemical-recurrence. Int J Cancer (2008) 122(10):2385-90 [PMID: 18214856].
2. John T, Caballero OL, Svobodová SJ, Kong A, Chua R, Browning J, Fortunato S, Deb S, Hsu M, Gedye CA, Davis ID, Altorki N, Simpson AJ, Chen YT, Monk M, Cebon JS. ECSA/DPPA2 is an embryo-cancer antigen that is coexpressed with cancer-testis antigens in non-small cell lung cancer. Clin Cancer Res (2008) 14(11):3291-8 [PMID: 18519755].

Signal Transduction

TGF-beta Program: Changing Paradigms

Cell signaling induced by transforming growth factor beta (TGFΒ) regulates normal cell processes such as proliferation, differentiation (the specialization of cell form and function) and apoptosis (programmed cell death), as well as the invasiveness and metastatic spread of cancer cells. The TGFΒ Program—a group of investigators from the LICR Brussels, Uppsala, Melbourne and Stockholm Branches plus LICR Affiliates and other collaborators—have been working together for nearly 10 years to further our understanding of TGFΒ signaling, and to explore how the molecules that transmit and respond to TGFΒ signals can be targeted to limit tumor growth and/or prevent metastasis.

A long-standing paradigm in TGFΒ signaling was that the signals are transmitted from the TGFΒ receptors on the cell’s surface to the cell’s nucleus via intracellular Smad proteins. In 2008, LICR Uppsala Branch researchers reported that TGFΒ triggers apoptosis through a new signaling mechanism that functions independently of Smads¹. A key component of this Smad-independent signaling is TRAF6, an intracellular protein that acts as a switch to prompt apoptosis. Future studies will determine if TRAF6 mediates TGFΒ responses other than apoptosis, such as cell proliferation or differentiation. This study was selected as a highlight in the top two weekly online resources for signal transduction researchers, Science Signaling and UCSD-Nature Signaling Gateway.

The team also discovered a new mechanism through which TGFΒ signaling is decreased or halted. The TGFΒ pathway is driven by the TGFΒ receptors’ kinase activity, which modifies signaling proteins inside the cell thereby activating those proteins. However, few other kinases have been identified as directly regulating TGFΒ signal transduction. The LICR Uppsala Branch investigators have now discovered that a kinase, SIK, cooperates with the inhibitory Smad7 protein to regulate the removal of TGFΒ receptor from the cell surface and its subsequent degradation inside the cell².

Finally, another line of research at the Uppsala Branch has cast new light on the epithelial-mesenchymal transition (EMT), a process by which some normal cells acquire the ability to migrate and invade tissues. In many tumors, TGFΒ signaling induces EMT. LICR investigators had previously identified HMGA2, a protein that regulates the expression of a number of genes, as a key determinant of TGFΒ-induced EMT. In a continuing exploration of this finding, the team has now elucidated some of the molecular mechanisms mediated by HMGA2 to cause EMT in breast cells³. In particular, the transcription factor SNAIL1, which is known to play a key role in tumor progression and EMT, was found to be regulated by HMGA2. The investigators were able to partially revert the EMT process by reducing the levels of SNAIL1, suggesting this protein might be a target for future therapeutic strategies.

References

1. Sorrentino A, Thakur N, Grimsby S, Marcusson A, von Bulow V, Schuster N, Zhang S, Heldin CH, Landström M. The type I TGF-beta receptor engages TRAF6 to activate TAK1 in a receptor kinase-independent manner. Nat Cell Biol (2008) 10(10):1199-207 [PMID: 18758450].
2. Kowanetz M, Lönn P, Vanlandewijck M, Kowanetz K, Heldin CH, Moustakas A. TGFbeta induces SIK to negatively regulate type I receptor kinase signaling. J Cell Biol (2008) 182(4):655-62 [PMID: 18725536].
3. Thuault S, Tan EJ, Peinado H, Cano A, Heldin CH, Moustakas A. HMGA2 and Smads co-regulate SNAIL1 expression during induction of epithelial-to-mesenchymal transition. J Biol Chem (2008) 283(48):33437-46 [PMID: 18832382].

 

Signal Transduction

Cytokines In Cancer and Myeloproliferative Diseases

Cytokines are cell-to-cell signaling molecules that regulate several physiological processes, including the immune and inflammatory responses. The binding of a cytokine to its receptor on the cell surface activates intracellular signaling cascades that transmit signals to the nucleus to alter gene expression. A breakdown in the integrity of these signaling pathways is thought to cause the development of cancers, myeloproliferative diseases (MPDs) and also some autoimmune disorders. LICR investigators further elucidated the molecular mechanisms causing dysregulated signaling through a crucial cytokine pathway, the intracellular JAK/STAT pathway.

Cytokine Signaling and Gastric Cancer

Gastric cancer is the second most common cause of cancer-related deaths around the world. While chronic stomach inflammation is a major risk factor for this cancer, the precise link between inflammation and cancer onset and progression has not yet been clarified. Persistent activation of the STAT3 protein is known to mediate inflammation-associated carcinogenesis in the stomach, and other tissues, but the exact mechanisms leading to STAT3 hyper-activation are also not well understood. Investigators at the LICR Melbourne Branch have now shown that overproduction of the cytokine IL-11 could be the main trigger for inflammation-associated gastric cancer¹. IL-11 activates intracellular STAT3 via the transmembrane gp130 receptor and associated JAK proteins. Using a mouse model of inflammation-dependent human gastric cancer, the team showed that genetic inhibitors of STAT1 and genetic and pharmacologic inhibitors of STAT3 blocked the IL-11 signaling pathway and reduced gastric inflammation and tumor formation. Cancer onset and progression in this mouse model shares striking similarities to human gastric tumor development and appearance, suggesting this model might be a powerful tool for pre-clinical screening of potential therapeutic strategies targeting STAT3 hyper-activation.

Characterization of JAK Mutations in Myeloproliferative Diseases

Hyperactive signaling through the JAK/STAT pathway is associated with the development of MPDs such as polycythemia vera (over production of red blood cells), essential thrombocythemia (excessive production of platelets) and idiopathic myelofibrosis (replacement of bone marrow with scar tissue). LICR investigators and others established several years ago that this dysregulation is the result of a specific amino acid substitution in the JAK2 enzyme—phenylalanine substituted for valine at amino acid residue position 617—which is known as V617F. This mutation is present in greater than 95% of polycythemia vera patients and 50% of the patients with essential thrombocythemia and idiopathic myelofibrosis. A team at the LICR Brussels Branch recently analyzed the physiological consequences of substituting other amino acids at the same location to identify additional mutations that may be present in MPDs. Although four potential amino acid substitutions that could induce constant signaling through the JAK/STAT pathway were found, only the amino acid tryptophan caused a level of activation similar to the V617F mutation identified in many MPDs². The team calculated that there is a very low probability of the valine being substituted by tryptophan in MPDs as this substitution requires three separate DNA mutations. In contrast, the V617F mutation results from a single DNA mutation. This work elucidated further the conformational changes in the JAK2 protein that lead to constitutive signaling, and the knowledge gained is being applied to the development of small molecule inhibitors that are specific enough to discriminate between the normal JAK2 and the V617F mutant form that causes MPDs.

References

1. Ernst M, Najdovska M, Grail D, Lundgren-May T, Buchert M, Tye H, Matthews VB, Armes J, Bhathal PS, Hughes NR, Marcusson EG, Karras JG, Na S, Sedgwick JD, Hertzog PJ, Jenkins BJ. STAT3 and STAT1 mediate IL-11-dependent and inflammation-associated gastric tumorigenesis in gp130 receptor mutant mice. J Clin Invest (2008) 118(5):1727-38 [PMID: 18431520].
2. Dusa A, Staerk J, Elliott J, Pecquet C, Poirel HA, Johnston JA, Constantinescu SN. Substitution of pseudokinase domain residue Val-617 by large non-polar amino acids causes activation of JAK2. J Biol Chem (2008) 283(19):12941-8 [PMID: 18326042].

Cancer Vaccine Research

While the immune system can recognize and target cancer cells, it is not always able to prevent tumor growth and disease progression. This could be because the immune response is insufficiently robust and/or sustained, and/or because the tumor has been able to suppress the immune response. LICR investigators are evaluating the ability of therapeutic cancer vaccines (TCVs) to induce or augment an immune response that is integrated (i.e. is comprised of antigen-specific antibodies plus antigen-specific CD4+ and CD8+ T cells) and can eliminate cancer cells. TCVs combine cancer antigens, which direct the immune system to selectively target cancer cells, with adjuvants, molecules that boost the immune response to the specific antigen. LICR clinical research teams, working within the Cancer Vaccine Collaborative, have been conducting clinical trials to measure the immune responses induced by different combinations of antigens and adjuvants, immunomodulators, and vaccine delivery methods. While TCVs are not yet a treatment option for cancer patients, multiple phase II and III clinical trials are currently testing the efficacy of this potential therapeutic modality.

NY-ESO-1 Vaccine Shows Potential for Bladder Cancer Patients

Immunotherapy with the tuberculosis vaccine BCG is highly successful in the treatment of superficial bladder cancer. However, there are no immunotherapies for patients with invasive bladder disease. In 2008, a team from the LICR New York Branch and M.D. Anderson Cancer Center in Houston (USA) reported the induction of antigen-specific antibody and T cell responses from the first TCV clinical trial in patients with bladder cancer¹. This phase I study demonstrated the safety and immunogenicity of recombinant NY-ESO-1 protein administered in combination with the adjuvants BCG and GM-CSF to a small population of patients with NY-ESO-1-expressing bladder cancers following surgical resection of the bladder. Specific antibody responses to NY-ESO-1 were induced in five of the six patients, and CD4+ T cell responses were observed in all six patients. However, CD8+ T cell responses were detected in only one patient. The team is now focusing on combining a NY-ESO-1 recombinant protein vaccine with other immunostimulatory agents to improve the frequency of CD8+ T cell responses. This study has expanded the number of tumor types potentially suitable for treatment with TCVs to include invasive bladder cancer.

Exploiting Immunological Memory for Cancer Vaccines

Immunological memory is established when a naïve T cell first encounters an antigen. That T cell gives rise to an antigen-specific T cell population that “remembers” the antigen; a very low level of the population remains in the body so that the immune system is primed to mount a rapid response should the antigen be encountered again. LICR teams are investigating whether a “boost” strategy—giving a cancer vaccine to establish memory, and then a booster immunization some time later—can induce a sustained, robust immune response against cancer cells. Clinical trials designed specifically to explore prime boost strategies for TCVs have been initiated by the Institute.

A team from the LICR Brussels Branch has been working on an intriguing hypothesis: that an immune response induced by a TCV, an ‘anti-vaccine response,’ might re-activate a spontaneous anti-tumor response that has been suppressed by the tumor. Melanoma patients vaccinated with MAGE-A3-based TCVs did mount MAGE-A3-specific CD8+ T cell responses, but the Brussels team found that the levels of the T cells were very low². A detailed analysis of the CD8+ T cell populations in two patients, both of whom showed some clinical response to TCVs, found that there were low levels of MAGE-A3-specific T cells in the blood and, surprisingly, an enrichment of T cells directed against MAGE-C2, another antigen, in the tumor. This study suggests several intriguing possibilities for understanding cancer immunology. The MAGE-A3 TCV might be acting to boost a primary immunization mounted spontaneously by the patient, or it might be reawakening anergic T cells

A 2004 clinical trial sponsored by LICR and conducted by investigators from the LICR New York Branch and Weill-Cornell Medical College in New York (USA) analyzed the immune response to a TCV based on the MAGE-A3 antigen in patients with non-small cell lung cancer (NSCLC). Some patients on this original study received the TCV with AS02B adjuvant added (TCV + AS02B) and some received the TCV without the adjuvant (TCV alone). In an extension of this trial, the team assessed whether the original immune responses could be boosted some years later³. Booster immunizations of TCV + AS02B given to seven patients who had originally received TCV + AS02B produced antigen-specific antibody levels at least equivalent to those observed after the initial vaccination given three years before. However, only two of the seven patients who received TCV alone in the initial vaccination were able to develop high levels of antibodies against MAGE-A3 following a booster TCV + AS02B immunization. The booster immunization also increased the number of CD4+ and CD8+ T cell populations recognizing different parts of the MAGE-A3 antigen in patients who originally received TCV + AS02B, which supports the LICR Brussels Branch hypothesis (above). This enhancement of immunological recognition did not occur in the patients who received TCV alone.

The finding that adjuvant is required in primary immunizations in order to induce a greater immune response contributed to the design of a MAGE-A3-based TCV currently in phase III trials. The pharmaceutical company GlaxoSmithKline (GSK) licensed the MAGE-A3 antigen from LICR and is testing the efficacy of the MAGE-A3 TCV, or “antigen-specific cancer immunotherapy” (ASCI), in the largest ever clinical trial for a lung cancer therapy. This trial is testing whether the MAGE-A3 ASCI can prevent the recurrence of NSCLC in patients whose tumor has been surgically resected.

Better Vaccines with Natural Peptides?

Some cancer vaccines are based on ‘analog’ peptides; synthetic peptides derived from the cancer antigen sequence, which have been modified to improve the efficiency of CTL recognition. These vaccines have been shown to produce strong T cell responses in patients. However, LICR Lausanne Branch investigators have discovered the responses induced by analog peptides to be sub-optimal for fighting tumors⁴. In the Lausanne Branch study, melanoma patients were vaccinated with an analog peptide or its corresponding natural peptide in combination with a powerful adjuvant, a molecule that boosts the immune response. The team found that immune responses induced by an analog peptide were less effective in recognizing and eradicating tumors; evidence for a rationale of developing cancer vaccines based on natural peptides.

References

1. 1. Sharma P, Bajorin DF, Jungbluth AA, Herr H, Old LJ, Gnjatic S. Immune responses detected in urothelial carcinoma patients after vaccination with NY-ESO-1 protein plus BCG and GM-CSF. J Immunother (2008) 31(9):849-57
2. 2. Carrasco J, Van Pel A, Neyns B, Lethé B, Brasseur F, Renkvist N, van der Bruggen P, van Baren N, Paulus R, Thielemans K, Boon T, Godelaine D. Vaccination of a melanoma patient with mature dendritic cells pulsed with MAGE-3 peptides triggers the activity of nonvaccine anti-tumor cells. J Immunol (2008) 180(5):3585-93
3. 3. Atanackovic D, Altorki NK, Cao Y, Ritter E, Ferrara CA, Ritter G, Hoffman EW, Bokemeyer C, Old LJ, Gnjatic S. Booster vaccination of cancer patients with MAGE-A3 protein reveals long-term immunological memory or tolerance depending on priming. Proc Natl Acad Sci USA (2008) 105(5):1650-5
4. 4. Speiser DE, Baumgaertner P, Voelter V, Devevre E, Barbey C, Rufer N, Romero P. Unmodified self antigen triggers human CD8 T cells with stronger tumor reactivity than altered antigen. Proc Natl Acad Sci USA (2008) 105(10):3849-54

Enzymatic Depletion

Cancer cells, by virtue of their accelerated rate of growth and proliferation, require a readily available supply of amino acids in order to create new proteins. LICR investigators are working with a collaborator to assess whether removing a required amino acid can halt cancer cell growth and proliferation.

Amino acids, the building blocks of proteins, are created by enzymatic reactions within cells. However, some cancers—including melanoma, and liver, pancreatic and prostate cancers—lack the enzyme arginosuccinate synthetase (ASS), which catalyzes the production of the amino acid arginine. To obtain this amino acid and synthesize proteins, cancer cells must scavenge arginine from the blood.

In combination with commercial partner Polaris Pharmaceuticals Inc., LICR is exploring whether depleting arginine from the blood is sufficient to cause cancer cell death by inhibiting protein synthesis. The candidate therapy is an enzyme, arginine deiminase (ADI), which catalyzes the irreversible conversion of arginine into citrulline, another amino acid. The depletion of arginine via ADI does not seem to affect normal cells that still have functional ASS and can thus create more arginine.

LICR is currently sponsoring and conducting a clinical trial assessing ADI safety and tumor response in patients with metastatic melanoma.

GM-CSF

The first discovery by LICR investigators to make it to commerical application was GM-CSF, a molecule that stimulates the production of white blood cells.

The innate immune system provides the first-line of defense against infectious organisms. Central to both the innate immune and inflammatory responses are the white blood cells, or leukocytes, a group of specialized cells that includes neutrophils, eosinophils, monocytes and macrophages. Leukocytes are produced when hematopoietic stem cells in the bone marrow differentiate into the specialized cell types in response to regulators, such as the colony-stimulating factors (CSFs). Many chemotherapeutic regimens destroy leukocytes, leaving oncology patients susceptible to infection and unable to undergo further chemotherapy treatments.

Investigators from the LICR Melbourne Branch and the Walter and Eliza Hall Institute of Medical Research (Australia) cloned GM-CSF (granulocyte/macrophage-CSF) in 1984. The first-in-man clinical trial of GM-CSF was conducted by the Melbourne Branch, and assessed the ability of GM-CSF to stimulate leukocyte production in cancer patients. The GM-CSF discovery was licensed to industrial partners to move its clinical development forward, and GM-CSF is now part of a treatment regimen that supports bone marrow transplantation and some chemotherapies.

A simplified diagram of blood cell differentiation. Specific growth factors (only some of which are depicted in green, below) control the differentiation of blood cell types from hematopoietic stem cells.



Clinical research uncovers two applications for human benefit

G250: Diagnosis and Therapy

Currently, patients with metastatic renal cell carcinoma (RCC) have a five year survival rate of approximately 30%. While new RCC chemotherapeutics are showing benefit in the clinic, therapeutic advances are still critically needed. Laboratory and clinical research conducted by LICR has led to the G250 antibody entering commercial drug development for both diagnosis and therapy of RCC.

The G250 antibody binds to the CAIX molecule, which is present on the surface of more than 85% of RCC cells, but is not present on normal kidney cells. Clinical trials sponsored and conducted by LICR determined that this monocloncal antibody is safe for patients, has a long half-life in the body and that shows excellent targeting to RCC tumors. Wilex AG (Munich, Germany), a biotechnology company with which LICR has had a long-standing research and development collaboration, is conducting phase III clinical trials to assess the potential of the G250 antibody as an adjuvant therapy, i.e. a therapy given after surgical removal of the tumor, in patients who have non-metastatic RCC. Wilex is developing the therapy application of G250 under the product name Rencarex®.

Another LICR-sponsored pilot trial assessing the use of G250 as a diagnostic tool for clear cell RCC, the most aggressive subtype of RCC, produced extraordinary results. The LICR team showed that using iodine-124 (124I)-labeled G250 antibody followed by positron emission tomography (PET) imaging, accurately identified clear cell RCCs with the specificity and positive predictive accuracy both at the 100% level. The development of a non-invasive diagnostic tool for clear cell RCC will make a substantial difference to the care and welfare of people suspected of having RCC, as they will not have to undergo surgery in order to differentiate between clear cell and non-clear cell RCC. Wilex AG is conducting a pivotal phase III trial to confirm radiolabeled G250 antibody is able to diagnose clear cell RCC with PET imaging. The diagnostic application of G250 is being developed under the product name Redectane®.

Specific targeting of clear cell renal cancer by I-124 cG250 immunoPET.The left image shows two lesions (solid and broken arrow) in the left kidney, evident on a CT scan.  ImmunoPET (middle image) shows radioactivity in only one of those lesions (solid arrow).  The right image is a superimposition (“fusion”) image, confirming radioactivity in the inferior lesion, which was the only one of the two to be of clear cell histology.