Nos recherches sont centrées sur l’immunologie tumorale, en particulier sur deux questions importantes en matière de développement de l’immunothérapie du cancer: (i) quels sont les mécanismes de production des antigènes tumoraux, et (ii) comment les tumeurs parviennent-elles souvent à résister à l’attaque immunitaire, et comment pouvons-nous empêcher ces résistances.

Les travaux effectués dans nos équipes au cours des années 1990 ont clairement établi l’existence sur les cellules cancéreuses de « marqueurs », appelés antigènes tumoraux, qui distinguent ces cellules cancéreuses des cellules normales de l’organisme et permettent  au système immunitaire de reconnaître les cancers comme des corps étrangers devant être détruits. Ceci a ouvert la voie à de nouvelles approches thérapeutiques visant à renforcer cette capacité du système immunitaire à rejeter les cancers. Certaines de ces approches sont  aujourd’hui entrées en pratique clinique courante, et permettent notamment de sauver des patients atteints de mélanome métastatique avancé. Ces nouvelles approches de thérapie anti-cancéreuse, appelées « immunothérapie du cancer » suscitent dès lors un intérêt croissant, à la fois parmi les cancérologues et au sein de l’industrie pharmaceutique. Cependant, malgré des succès cliniques indiscutables, ces approches d’immunothérapie sont actuellement limitées par deux phénomènes. Le premier est l’induction de phénomènes toxiques de nature autoimmunitaire, liés au fait que dans certaines approches d’immunothérapie la stimulation de l’immunité anti-tumorale s’accompagne d’une stimulation de l’autoimmunité, c’est-à-dire une attaque des tissus sains par le système immunitaire. La deuxième limitation est liée au fait que beaucoup de cancers, au cours de leur évolution progressive, acquièrent la capacité de résister à l’attaque immunitaire. Nos recherches actuelles visent à comprendre ces deux obstacles et à développer des stratégies thérapeutiques permettant de les contourner. Le premier obstacle est abordé par une étude approfondie des mécanismes de production des antigènes tumoraux, qui permet de définir quels sont les antigènes qui sont les plus spécifiques des tumeurs, et donc de mieux choisir ceux qui devront être ciblés préférentiellement pour obtenir des réponses immunitaires anti-cancéreuses dépourvues de toxicité autoimmunitaire. Le deuxième obstacle est abordé par l’étude détaillée du microenvironnement tumoral, dont la nature immunosuppressive semble clairement limiter l’efficacité de l’immunothérapie. Différentes stratégies thérapeutiques sont développées pour combattre cette immunosuppression.

 

  •  Mécanismes de production des antigènes tumoraux

Les antigènes tumoraux sont principalement reconnus par des globules blancs spécialisés appelés lymphocytes T cytotoxiques (CTL). Ceux-ci reconnaissent à la surface des cellules cancéreuses des petits morceaux de protéines appelés peptides, composés de huit à dix acides aminés, qui proviennent de protéines intracellulaires et sont présentés à la surface cellulaire par des molécules de classe I du complexe majeur d’histocompatibilité (MHC, également appelé HLA chez l’homme). Les peptides sont habituellement issus de la dégradation des protéines intracellulaires par le protéasome, une particule protéolytique localisée dans le cytoplasme et le noyau. Les peptides sont ensuite transportés vers la surface cellulaire après association avec des molécules MHC. Nos recherches ont montré qu’il existe différentes formes de protéasome, et que celles-ci différaient quant à leur capacité à produire les peptides correspondant aux antigènes tumoraux. Il en résulte que les antigènes présentés à la surface des cellules cancéreuses dépendent en partie de la composition en protéasome de ces cellules. Nous étudions en détail ces phénomènes de manière à améliorer le choix des antigènes à cibler pour l’immunothérapie. Nous étudions aussi une nouvelle fonction du protéasome, que nous avons découverte, qui permet l’épissage de peptides, à savoir la production de peptides à partir de fragments non contigus dans la protéine parentale, selon un mode « cut and paste ».

Nous étudions également comment certains cancers tentent d’échapper au rejet immunitaire en « se cachant », c’est-à-dire en perdant l’expression des antigènes tumoraux qui les « dénoncent » au système immunitaire. Ce faisant, ils démasquent d’autres antigènes que nous tentons de caractériser afin de mieux combattre les cancers, dans ce qui devient une sorte de jeu d’échecs avec le cancer.

 

  •  Combattre l’immunosuppression locale induite par la tumeur

Il devient clair à présent que lorsque l’immunothérapie est inefficace chez certains patients cancéreux, c’est souvent lié à la présence de mécanismes immunosuppresseurs au niveau de la tumeur, qui empêchent le bon fonctionnement du système immunitaire. Nous étudions ces mécanismes dans des modèles murins, notamment un modèle transgénique de mélanome inductible que nous avons créé, dans lequel nous avons trouvé que les tumeurs induisent sélectivement la mort par apoptose des lymphocytes qui viennent pour les détruire. Nous étudions les mécanismes moléculaires responsables de ces phénomènes d’immunosuppression. Nous avons déjà montré qu’un de ces mécanismes se base sur la dégradation d’un acide aminé dont les lymphocytes ont un besoin impérieux, le tryptophane.Les tumeurs expriment un enzyme, appelé indoleamine dioxygenase (IDO) qui dégrade rapidement le tryptophane et paralyse ainsi le système immunitaire. Dans le contexte d’une spin-off que nous avons fondée en 2012, appelée iTeos Therapeutics, nous recherchons des inhibiteurs de l’IDO qui pourraient être utilisés en thérapie anti-cancéreuse. Nous tentons aussi de comprendre les mécanismes responsables de l’expression de cet enzyme par les cancers.

Nous avons aussi découvert d’autres mécanismes d’immunosuppression dont nous étudions les acteurs moléculaires afin de les combattre.

In recent years, cancer immunotherapy made tremendous progresses and reached the clinical arena by showing its ability to prolong the survival of advanced cancer patients. This was largely based on the discovery of tumor antigens by our groups in Brussels in the nineties, a finding that demonstrated that our immune system has the capacity to recognize cancer cells as foreign bodies. These recent clinical results have generated enormous interest in the oncology field and the pharmacological industry. Yet, clinical benefits remain limited to a subset of patients, and further research is needed to understand the reason for this. Our current research focuses on two aspects that are relevant to this question. The first is the processing of tumor antigens, i.e. the intracellular mechanisms responsible for the expression of antigens at the surface of tumor cells. The second is the tumor microenvironment, whose immunosuppressive properties emerge as a major reason why many patients currently do not benefit from cancer immunotherapy.

 

I. PROCESSING OF TUMOR ANTIGENS

1. Peptide splicing by the proteasome 
N. Vigneron, V. Stroobant, A. Michaux

Tumor antigens relevant for cancer immunotherapy consist of peptides presented by MHC class I molecules and derived from intracellular tumor proteins. They result from degradation of these proteins, mainly exerted by the proteasome. We have identified a new mode of production of antigenic peptides, which involves the splicing of peptide fragments by the proteasome (1). Peptide splicing occurs in the proteasome catalytic chamber through a reaction of transpeptidation involving an acyl-enzyme intermediate (Figure 1). Splicing of peptide fragments can occur in the forward or reverse order to that in which they appear in the parental protein (2). We have now described five spliced peptides, three of which are spliced in the reverse order (3, 4) One of these peptides also contains two additional post-translational modifications, resulting in the conversion of asparagines into aspartic acids, through a process a N-glycosylation/deglycosylation (3). We also showed that the splicing reaction required a minimal size of 3 amino acids for the fragments to splice (4). Both the standard proteasome and the immunoproteasome have the ability to splice peptides. However, their ability to produce a given spliced peptide varies according to their ability to perform the relevant cleavages to liberate the fragments to splice.

 

 

Legend: Model of the peptide-splicing reaction in the proteasome. The active site of the catalytic subunits of the proteasome is made up of the side-chain of a threonine residue, which initiates proteolysis by performing a nucleophilic attack on the carbonyl group of the peptide bond. An acyl-enzyme intermediate is formed, which is then liberated by hydrolysis. In the peptide-splicing reaction, a second peptide fragment appears to compete with water molecules for performing a nucleophilic attack on the acyl-enzyme intermediate, resulting in a transpeptidation reaction producing the spliced peptide. Experimental support for this model of reverse proteolysis includes evidence that the energy required to create the new peptide bond is recovered from the peptide bond that is cleaved at the amino-terminus of the excised fragment, and that the amino-terminus of the other fragment needs to be free for transpeptidation to occur.

 

2. Intermediate proteasome types
J. Abi Habib, E. De Plaen, B. Guillaume, N. Vigneron, A. Michaux

The proteasome exists in two forms: the standard proteasome, which is constitutively present in most cells, and the immunoproteasome, which is constitutive in many immune cells and can be induced by interferon-gamma in most other cells. They differ by the three catalytic subunits they use: ß1, ß2 and ß5 for the standard proteasome; ß1i, ß2i and ß5i for the immunoproteasome. We have described two new proteasome subtypes that are intermediate between the standard proteasome and the immunoproteasome (5). They contain only one (ß5i) or two (ß1i and ß5i) of the three inducible catalytic subunits of the immunoproteasome. These intermediate proteasomes represent 30 to 54% of the proteasome content of human liver, colon, small intestine and kidney. They are also present in human tumor cells and dendritic cells. They uniquely process several tumor antigens (5, 6). We are currently studying the function of these intermediate proteasomes, not only in terms of processing of antigenic peptides, but also for other functional aspects in which the proteasome plays a crucial role, such as the regulation of the cell cycle, the activation of transcription factors and the regulation of inflammation and immune responses.

 

3. Other proteases involved in antigen processing
N. Vigneron, A. Michaux, V. Stroobant

We are interested in characterizing the processing of human antigenic peptides that are not produced by the proteasome. We studied a proteasome-independent peptide derived from tumor protein MAGE-A3, and identified insulin-degrading enzyme as the protease producing this peptide (7). Insulin-degrading enzyme is a cytosolic metallopeptidase not previously known to play a role in the antigen processing pathway. The parental protein MAGE-A3 appears to be degraded along two parallel pathways involving insulin-degrading enzyme or the proteasome, each pathway producing a distinct set of antigenic peptides presented by MHC class I molecules. We are currently studying the processing of other proteasome-independent peptides and aiming to identify the protease(s) involved.

 

4. TAP-independent antigenic peptides
N. Vigneron, V. Stroobant, L. Pilotte

Presentation of most peptides depends on TAP, which transports peptides from the cytosol to the endoplasmic reticulum. A number of viruses and tumor cells tend to reduce their TAP expression to escape immune recognition. Therefore, there is great interest in the potential therapeutic use of peptides that are still presented in the absence of TAP. We are studying several such tumor peptides derived from cytosolic proteins. We aim at characterizing their processing and identifying the alternative transporter in charge of their transfer from the cytosol to the endoplasmic reticulum.

 

5. Cross-presentation
W. Ma, N. Vigneron, in collaboration with P. Courtoy and P. Van Der Smissen

Class I and class II molecules of the Major Histocompatibility Complex (MHC) are responsible for the presentation of antigenic peptides derived from intracellular proteins or from engulfed exogenous proteins, respectively. As an exception to this rule, cross-presentation enables dendritic cells to present on their MHC class I molecules antigenic peptides derived from exogenous material, through a mechanism that remains unclear. Cross-presentation is essential to the activation of CD8+ T lymphocytes against antigens derived from tumors and from viruses that do not infect dendritic cells. It is particularly efficient with long peptides, which are used in cancer vaccines. We studied the mechanism of long-peptide cross-presentation using human dendritic cells and specific CTL clones against melanoma antigens gp100 and Melan-A/MART1. We found that long-peptide cross-presentation does not depend on the proteasome nor the TAP transporter, and therefore follows a vacuolar pathway. We also observed that it makes use of newly synthesized MHC class I molecules that are loaded with suboptimal peptides. These nascent MHC-I molecules appear to diverge from the classical secretion pathway at an early stage and reach the late endosomes, where they exchange their suboptimal peptide cargo for the cross-presented peptide before reaching the cell surface in an endoH-sensitive form. These results indicate an alternative secretion pathway followed by HLA-I molecules that are used for cross-presentation, and may have implications for the development of vaccines based on long peptides.

 

II. MECHANISMS OF TUMORAL IMMUNE RESISTANCE

1. Indoleamine 2,3-dioxygenase
M. Hennequart, J. Lamy, E. De Plaen, L. Pilotte, V. Stroobant, D. Colau

We previously discovered that tumors often resist immune rejection by expressing Indoleamine 2,3-dioxygenase (IDO), a tryptophan-degrading enzyme that is profoundly immunosuppressive (8). We showed that immune rejection was restored by administration of a pharmacological inhibitor of IDO. In collaboration with medicinal chemists in Namur and Lausanne, we identified several families of new IDO inhibitors that will be further optimized to develop drug candidates. We currently pursue functional studies on the mechanisms of IDO-induced immunosuppression, and on the signaling pathway responsible for IDO expression in tumors.

 

2. Tryptophan-dioxygenase
F. Schramme, D. Hoffmann, S. Klaessens, L. Pilotte, J. Lamy, E. De Plaen, V. Stroobant, D. Colau

Besides IDO, we recently uncovered the role of tryptophan-dioxygenase (TDO) in tumoral immune resistance (9). TDO is an unrelated tryptophan-degrading enzyme, which is highly expressed in the liver to regulate systemic tryptophan levels. We found TDO to be expressed in a high proportion of human tumors. We showed that TDO-expressing mouse tumors are no longer rejected by immunized mice. Moreover, we developed a TDO inhibitor, which, upon systemic treatment, restored the ability of mice to reject tumors (9). These results describe a mechanism of tumoral immune resistance based on TDO expression and establish proof-of-concept for the use of TDO inhibitors in cancer therapy. In April 2012, we have launched an LICR spin-off company, iTeos Therapeutics, which will develop inhibitors of IDO and TDO.

 

3. Transgenic mice developing autochthonous melanomas expressing P1A
C. Powis de Tenbossche, S. Cané, J. Zhu, C. Uyttenhove, N. Arts, E. De Plaen in collaboration with J. Van Snick and C. Uyttenhove

We have created a mouse model of autochthonous inducible melanoma expressing a defined tumor antigen (TIRP10B) (10). In this model, melanomas are induced (70% incidence) with tamoxifen, which, by activating CreER in melanocytes, induces the expression of Ha-Ras, the deletion of INK4a/ARF and the expression the tumor antigen encoded by cancer/germline gene P1A. A unique feature of this model is that melanomas first develop as non-aggressive highly pigmented tumors (Mela), which later dedifferentiate into unpigmented highly aggressive inflammatory tumors (Amela). We found that TGF-ß was a key factor responsible for this switch to aggressive tumors, which is reminiscent of the epithelial-to-mesenchymal transition (EMT) described in other contexts. We developed antibodies able to neutralize TGFß1 and TGFß3, and found that the former were able to increase survival of mice in this melanoma model. These results support the use of TGFß neutralizing therapies in the treatment of human melanoma.

The loss of pigmentation in aggressive tumors appears to result from the strong inflammation, and we identified a microRNA that is induced by interleukin-1 and downregulates expression of MITF, a transcription factor acting as a master regulator of pigmentation.

In this model, both pigmented (Mela) and unpigmented (Amela) tumors express the tumor antigen encoded by P1A. Mela tumors are ignored by the immune system, while Amela tumors are infiltrated by T lymphocytes that are rendered ineffective. We are studying the mechanisms responsible for this ineffectiveness. Our current results indicate that the tumor microenvironment actively induces the apoptosis of tumor-specific T lymphocytes that infiltrate the tumor. We are studying the molecular mechanisms responsible for this apoptosis.

 

References

  1. N. Vigneron, V. Stroobant, J. Chapiro, A. Ooms, G. Degiovanni, S. Morel, P. van der Bruggen, T. Boon, B. Van den Eynde, An antigenic peptide produced by peptide splicing in the proteasome. Science, 2004, 304, 587-590
  2. E. H. Warren, N. J. Vigneron, M. A. Gavin, P. G. Coulie, V. Stroobant, A. Dalet, S. S. Tykodi, S. M. Xuereb, J. K. Mito, S. R. Riddell, B. J. Van den Eynde, An antigen produced by splicing of noncontiguous peptides in the reverse order. Science, 2006, 313, 1444-1447
  3. A. Dalet, P. F. Robbins, V. Stroobant, N. Vigneron, Y. F. Li, M. El-Gamil, K. Hanada, J. C. Yang, S. A. Rosenberg, B. J. Van den Eynde, An antigenic peptide produced by reverse splicing and double asparagine deamidation. Proc. Natl. Acad. Sci. U.S.A., 2011, 108, E323-331
  4. A. Michaux, P. Larrieu, V. Stroobant, J. F. Fonteneau, F. Jotereau, B. J. Van den Eynde, A. Moreau-Aubry, N. Vigneron, A spliced antigenic peptide comprising a single spliced amino acid is produced in the proteasome by reverse splicing of a longer peptide fragment followed by trimming. J. Immunol., 2014, 192, 1962-1971
  5. B. Guillaume, J. Chapiro, V. Stroobant, D. Colau, B. Van Holle, G. Parvizi, M. P. Bousquet-Dubouch, I. Theate, N. Parmentier, B. J. Van den Eynde, Two abundant proteasome subtypes that uniquely process some antigens presented by HLA class I molecules. Proc. Natl. Acad. Sci. USA, 2010, 107, 18599-18604
  6. B. Guillaume, V. Stroobant, M. P. Bousquet-Dubouch, D. Colau, J. Chapiro, N. Parmentier, A. Dalet, B. J. Van den Eynde, Analysis of the processing of seven human tumor antigens by intermediate proteasomes. J. Immunol., 2012, 189, 3538-3547
  7. N. Parmentier, V. Stroobant, D. Colau, P. de Diesbach, S. Morel, J. Chapiro, P. van Endert, B. J. Van den Eynde, Production of an antigenic peptide by insulin-degrading enzyme. Nat. Immunol., 2010, 11, 449-454
  8. C. Uyttenhove, L. Pilotte, I. Theate, V. Stroobant, D. Colau, N. Parmentier, T. Boon, B. J. Van den Eynde, Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Nat. Med., 2003, 9, 1269-1274
  9. L. Pilotte, P. Larrieu, V. Stroobant, D. Colau, E. Dolusic, R. Frederick, E. De Plaen, C. Uyttenhove, J. Wouters, B. Masereel, B. J. Van den Eynde, Reversal of tumoral immune resistance by inhibition of tryptophan 2,3-dioxygenase. Proc. Natl. Acad. Sci. U.S.A., 2012, 109, 2497-2502
  10. I. J. Huijbers, P. Krimpenfort, P. Chomez, M. A. van der Valk, J. Y. Song, E. M. Inderberg-Suso, A. M. Schmitt-Verhulst, A. Berns, B. J. Van den Eynde, An inducible mouse model of melanoma expressing a defined tumor antigen. Cancer Res., 2006, 66, 3278-3286

 

Complete list on PubMed
Benoît Van den Eynde
Institut de Duve
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