Project Funding Details

Background: Following the encouraging clinical responses observed in cancer patients treated with anti-CTLA-4, -PD-1, and -PD-L1antibodies, immunotherapy shows great promise for the treatment of cancer1-5. The blockade of co-inhibitory pathways in T-cells promotes their activation and triggers anti-tumour immunity. The latter was shown to be driven against tumour-mutated antigens (neo-antigens) and to be dependent on the existence of neoantigen- specific, activated T-cells, prior to therapeutic intervention6-9. This observation suggests the complementary enhancement of T-cell responses by means of neo-antigen vaccination and/or adoptive transfer of neo-antigen-specific T-cell clones. The accumulated evidence on an association between the occurrence of natural antitumour immune responses in colorectal cancers (CRCs) and improved clinical prognosis, makes CRC patients excellent candidates to benefit from immunotherapy10-12. General aim: To demonstrate that CRC patients can benefit from personalized, neo-antigentargeted therapies. Plan of investigation and intended results: I will screen the coding genomes of advanced CRCs (stage III and IV) by whole-exome and RNA next-generation sequencing (NGS). Somatic mutation profiles (mutanomes) will be annotated and neo-antigens corresponding to the transcribed mutations will be tested for their ability to induce activation of autologous Tcells derived from tumour infiltrating lymphocytes (TILs) and peripheral blood13-15. The discovery of neo-antigen-specific T-cell clones in CRC patients would support the development of anti-cancer therapies consisting of neo-antigen-based vaccines and/or adoptive transfer of neo-antigen-specific T-cell clones (specific aim 1). By performing RNA sequencing, the detection of fusion and frame-shifted transcripts is also made possible. I will investigate whether the previous generate neo-antigens with increased immunogenicity when compared to antigens resulting from single amino acid substitutions (specific aim 2). By interrogating T-cells that are both derived from TILs and from blood, I will determine whether neo-antigen-specific clones are being supressed from entering tumour tissues16. To correlate these findings with the tumours? microenvironment, a high-throughput, phenotypical analysis of tumour-infiltrating immune cells will be performed by mass cytometry in the same tissues that will undergo neo-antigen screening17. The occurrence or lack of neo-antigen recognition by autologous TILs can then be correlated to the composition of the tumours? immune cell infiltrate and support the discovery of biomarkers that associate with treatment responses to immunotherapy (specific aim 3). CRCs can be divided into mismatch repair-proficient (0.5?5 mutations/Mb, ~85% of all cases) and mismatch repair-deficient (10?500 mutations/Mb, ~15% of all cases)18. Mismatch repair-deficient tumours are prime candidates for immunotherapeutic intervention due to their high mutational load and thus, immunogenic character6-9,19,20. Nevertheless, Human Leukocyte Antigen (HLA) class I expression, an essential condition for neo-antigen recognition by CD8+ T-cells21, is lost in approximately 60% of the mismatch repair-deficient tumours, which may frustrate T-cell-mediated immunotherapies22,23. On the other hand, HLA class I expression is maintained in at least two-thirds of mismatch repair-proficient cases and in up to 40% of mismatch repair-deficient tumours, which means that the majority of CRC patients are eligible for T-cell-based immunotherapies22,23. In light of this, neo-antigen screening will only be performed in HLA class I-positive tumours. Furthermore, two additional (sub-)aims will be pursued: 1) To determine whether mismatch repair-proficient CRCs, with lower mutations rates, can benefit from neo-antigen-targeted therapies (under specific aim 1). The presence of only few tumour neo-antigens might preclude the initiation of natural anti-tumour immune responses. On the other hand, the ?artificial? induction of T-cell responses against few neoantigens could disturb the immune?s system ?steady-state? and tip the balance towards antitumour immunity. Mismatch repair-proficient CRCs are particularly interesting for immunotherapeutic intervention since immune evasive phenotypes such as aberrant HLA class I expression are less frequent in these tumours22-25. 2) To unravel the mechanisms of immune escape that explain the persistence of HLA class I-positive, mismatch repair-deficient tumours with high mutational loads. This will be achieved by performing deep immunophenotyping in both HLA class I-positive and ?negative mismatch repair-deficient tumours (specific aim 4). Relevance for cancer research and patient care: Over one million CRC cases are diagnosed every year, making it the fourth most common malignancy and one of the most frequent causes of cancer-related deaths worldwide26. The current proposal is aimed at implementing neo-antigen-targeted, personalized immunotherapies for CRC treatment. CRC patients are excellent candidates to benefit from immunotherapy and considerable improvements in patient survival are to be expected in a clinical translation of the current proposal. Furthermore, the tumour-specific character of the treatment is unlikely to produce a negative impact in the quality of life of patients. Although the current proposal is focused on CRC, a similar approach can be applied to most cancer types and therefore, the social and scientific merit of the current proposal has a wide reach. Implementation: The results derived from my proposal will be communicated in international scientific journals as well as in national and international conferences. The potential of applying personalized immunotherapy for the treatment of advanced CRCs will be communicated to the lay press and patient organizations. This will be essential to strengthen patients? perception that high-quality, translational research is being undertaken. I anticipate that clinical trials with CRC patients, based on the current proposal, can be initiated after the last year of its implementation. Collaborations: This proposal employs a multidisciplinary approach, where collaborations that were established play a fundamental role in complementing my expertise in nextgeneration sequencing analysis of cancer genomes27,28 and in CRC tumor immunology10,22,29,30. Collaborators and respective expertise: ? Prof.dr. S. van der Burg and Dr. Els Verdegaal (Experimental Clinical Oncology, LUMC) ? T-cell-based immunotherapies. ? Prof.dr. Qiang-Pan Hammarström (department of Laboratory Medicine, Karolinska Institute, Sweden) ? Profiling of T-cell receptor repertoires. ? Prof.dr. Frits Koning (Immunohaematology and Blood Transfusion, LUMC) ? Immunophenotyping by mass cytometry. ? Dr. Ekaterina Jordanova (Center For Gynecological Oncology Amsterdam) - Immunophenotyping of the tumour?s microenvironment. ? Prof.dr. Peter ten Dijke (Molecular Cell Biology, LUMC) ? TGF? signalling in malignancies. ? Prof.dr. Hans Morreau, Dr. Tom van Wezel, and Dr. Dina Ruano (Department of Pathology, LUMC) ? Colorectal cancer pathology and genetics. ? Prof.dr. Hendrick-Jan Guchelaar and Dr. Maarten Zandvliet (Clinical Pharmacology and Toxicology, LUMC) ? Development of GMP-grade immunotherapeutic applications. ? Prof.dr. Kris Thielemans (Laboratory of Molecular & Cellular Therapy, Vrije Universiteit Brussel) ? Translation of experimental immunotherapies to clinical applications.

Aanleiding Elk jaar worden alleen in Nederland al 13.000 mensen gediagnosticeerd met dikkedarmkanker en sterven er rond de 5000 mensen aan deze ziekte. De maatschappelijke gevolgen veroorzaakt door deze ziekte vragen om ontwikkeling van innovatieve en effectieve therapeutische strategieën. Vroegtijdige diagnose van de ziekte vergroot de kans op volledige genezing en het belang hiervan wordt onderstreept door de invoering van het landelijk bevolkingsonderzoek. Bij dit onderzoek wordt de ontlasting van iedereen tussen de 55 en 75 wordt onderzocht op bloedsporen. Dit laatste kan wijzen op de aanwezigheid van darmpoliepen (de voorlopers van kanker), of erger: een kwaadaardig proces. Ondanks vroege opsporing zal bij een groot aantal individuen de ziekte nog terugkomen of pas in een vergevorderd stadium ontdekt worden. Dit zullen ook individuen onder de 55 jaar zijn, die nog niet voor een preventief onderzoek in aanmerking kwamen. Doel Het huidige voorstel heeft als doel te onderzoeken of patiënten met vergevorderde dikkedarmkanker baat hebben bij stimulatie van het natuurlijk aanwezige afweersysteem tegen ziekten, het immuunsysteem. Verwacht wordt dat deze behandeling zal leiden tot aanzienlijke verbetering in de kans op ziektevrije overleving en genezing in dikkedarmkanker patiënten. In een aantal andere ziekten, zoals kwaadaardige melanomen en sommige vormen van longkanker, blijkt deze strategie goed te werken. Aanpak Met de meest moderne DNA technologie zullen foutjes in de DNA code van dikke darmtumoren in kaart worden gebracht. Op basis van DNA worden uiteindelijk eiwitten gemaakt, de functionele bouwstenen in een cel. Veranderingen in het DNA leiden tot veranderde eiwitten, die als onnatuurlijk worden gezien door het lichaamseigen afweersysteem. In de tumor wordt de natuurlijke afweer geremd, door blokkades in de afweerreactie of door de samenstelling van tumor-specifieke afweercellen. In dit project zullen die tumor-specifieke afweercellen worden gekweekt en getest worden op remmende factoren of remmende samenstelling. Bovendien zullen geselecteerde tumoreiwitten in gezuiverde vorm en in hoge concentratie worden teruggegeven aan de gekweekte afweercellen, met als doel deze afweercellen te activeren en op deze manier een anti-tumor reactie op te wekken. Belang voor de patiënt Stimulatie van een lichaamseigen anti-tumor reactie is momenteel één van de meestbelovende opties om vergevorderde vormen van kanker aan te pakken. Dit voorstel richt zich op de maatschappelijke toepassing, en resultaten van dit onderzoek kunnen zorgen voor ondersteuning van klinische trials met het vermijden van ongewenste bijwerkingen. De gekozen methoden zullen bij succes eveneens toepasbaar zijn bij andere soorten van kan