Source: http://ludwigcancerresearch.org/location/brussels-branch/benoit-j-van-den-eynde-lab
Timestamp: 2019-04-23 02:23:03+00:00

Document:
• What are the mechanisms of production of tumor antigens?
• How do many tumors manage to resist immune rejection and what can we do about it?
Main interest is tumor genetics—genes that are induced or repressed specifically in tumor cells and that enable these cells to escape the immune response. One example is the repression of melanocyte differentiation genes encoding tumor antigens in melanoma cells in the presence of IL-1ß.
Research into the immunobiological and cell biological features of dendritic cells that modulate their ability to cross-present exogenous antigens, so as to give better instructions in dendritic cell based immunotherapy of tumor or infection diseases in clinic.
I bring my expertise in mass spectrometry, analytical science and peptide chemistry to the development and implementation of bioanalytical methods. In collaborative research, these tools contribute to the identification of new tumor antigens and provide a deeper understanding of the molecular mechanisms involved in the processing of tumor antigens and tumoral immune resistance.
As a post-doctoral researcher in the laboratory of tumor immunology and antigen processing, my work mostly focuses on understanding the parameters involved in the production of antigenic peptides recognized by cytolytic T lymphocytes on tumors, to define which peptides are best suited for the development of anti-cancer vaccine. My main interest lies in the role of proteasome, which is the main complex responsible for the release of antigenic peptides from the degradation of intracellular proteins. Additionally, I study the importance of TAP and tapasin in the processing of antigenic peptides as these two proteins often down-regulated in tumors.
I am seeking to identity and understand the mechanisms of the local immunosuppression and extracellular matrix remodeling in a mouse model of inducible melanoma, referred as TirRP-10B;Ink4a/Arfflox/flox;H-2d/d, which recapitulates the natural interaction and cross-talk between the tumor, the stroma and the immune system. Preliminary gene expression analysis revealed a clear TGFβ signature occurring in the most aggressive form of tumor (not-pigmented) as compared to the less aggressive (pigmented) form. Thus, employing newly generated TGFβ neutralizing antibodies we would like to understand whether TGFβ neutralization retards/prevents EMT-like dedifferentiation and thereby reduces tumor progression.
My group studies the immune responses to cancer, with the long-term goal of harnessing the immune system to fight cancer. We contributed to the molecular definition of human tumor antigens, which has opened a new era in cancer immunotherapy, recently showing its first clinical applications in the treatment of melanoma. We now follow two lines of research. The first focuses on the processing of tumor antigens, studying the role of the proteasome and other proteases in the production of tumor antigenic peptides. The second studies innovative preclinical models for cancer immunotherapy to decipher the mechanisms whereby tumors resist immune rejection. The long-term objectives are to better understand the interaction of tumors with the immune system and devise strategies to improve the efficacy of cancer immunotherapy.
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 described four spliced peptides, two 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 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.5 We are working on additional examples of spliced antigenic peptides.
Figure 1. 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.
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.6 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.6,7 We are 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.
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.8 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 studying the processing of other proteasome-independent peptides and aiming to identify the protease(s) involved.
Presentation of most peptides depends on TAP, which transports peptides from the cytosol to the ER. 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 to characterize their processing and identify the alternative transporter in charge of their transfer from the cytosol to the endoplasmic reticulum.
Cross-presentation is the pathway whereby endocytosed proteins can be presented on MHC class-I molecules. This process, which is active in dendritic cells, has not been worked out in detail. Several models have been proposed. One involves the transfer of the endocytosed antigen from the endosome to the cytosol, where it follows the classical MHC class I processing pathway involving the proteasome and TAP. Another model, called the vacuolar pathway involves processing of the antigen inside the endocytic compartment and binding of the peptides to MHC class I molecules recycled from the cell surface in recycling vesicles. We have set up a model system based on human dendritic cells to study in vitro cross-presentation of human tumor antigens.
1. Vigneron, N., V. Stroobant, J. Chapiro, A. Ooms, G. Degiovanni, S. Morel, P. van der Bruggen, T. Boon, and B. Van den Eynde. 2004. An antigenic peptide produced by peptide splicing in the proteasome. Science 304:587-590.
2. Warren, E.H., 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, and B.J. Van den Eynde. 2006. An antigen produced by splicing of noncontiguous peptides in the reverse order. Science 313:1444-1447.
3. Dalet, A., P.F. Robbins, V. Stroobant, N. Vigneron, Y.F. Li, M. El-Gamil, K. Hanada, J.C. Yang, S.A. Rosenberg, and B.J. Van den Eynde. 2011. An antigenic peptide produced by reverse splicing and double asparagine deamidation. Proc. Natl. Acad. Sci. U.S.A. 108:E323-331.
4. Dalet, A., N. Vigneron, V. Stroobant, K. Hanada, and B.J. Van den Eynde. 2010. Splicing of distant peptide fragments occurs in the proteasome by transpeptidation and produces the spliced antigenic peptide derived from fibroblast growth factor-5. J. Immunol. 184:3016-3024.
5. Dalet, A., V. Stroobant, N. Vigneron, and B.J. Van den Eynde. 2011. Differences in the production of spliced antigenic peptides by the standard proteasome and the immunoproteasome. Eur. J. Immunol. 41:39-46.
6. Guillaume, B., J. Chapiro, V. Stroobant, D. Colau, B. Van Holle, G. Parvizi, M.P. Bousquet-Dubouch, I. Theate, N. Parmentier, and B.J. Van den Eynde. 2010. Two abundant proteasome subtypes that uniquely process some antigens presented by HLA class I molecules. Proc. Natl. Acad. Sci. USA 107:18599-18604.
7. Guillaume, B., V. Stroobant, M.P. Bousquet-Dubouch, D. Colau, J. Chapiro, N. Parmentier, A. Dalet, and B.J. Van den Eynde. 2012. Analysis of the processing of seven human tumor antigens by intermediate proteasomes. J. Immunol. 189:3538-3547.
8. Parmentier, N., V. Stroobant, D. Colau, P. de Diesbach, S. Morel, J. Chapiro, P. van Endert, and B.J. Van den Eynde. 2010. Production of an antigenic peptide by insulin-degrading enzyme. Nat. Immunol. 11:449-454.
We previously discovered that tumors often resist immune rejection by expressing Indoleamine 2,3-dioxygenase (IDO), a tryptophan-degrading enzyme that is profoundly immunosuppressive.9 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.10,11,12,13 We are pursuing functional studies on the mechanisms of IDO-induced immunosuppression, and on the signaling pathway responsible for IDO expression in tumors.
Besides IDO, we recently uncovered the role of tryptophan-dioxygenase (TDO) in tumoral immune resistance.14 TDO is an unrelated tryptophan-degrading enzyme that 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 that, upon systemic treatment, restored the ability of mice to reject tumors.14,15 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 launched a Ludwig spin-off company, iTeos Therapeutics, to develop inhibitors of IDO and TDO.
We have created a mouse model of autochthonous inducible melanoma expressing a defined tumor antigen (TIRP10B).16 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 de-differentiate into unpigmented highly aggressive inflammatory tumors (Amela). The mechanisms that trigger this phenotype shift are unknown. Both 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.17 This is accompanied with exacerbated systemic inflammation, involving disruption of secondary lymphoid organs, extramedullary hematopoiesis and accumulation of immature myeloid cells. Although more difficult to study than classical models based on transplantable tumors, we believe this model is more relevant to the human situation as it recapitulates the long-lasting tumor-host relationship that results in tumor tolerance. In addition, melanoma shows a similar biphasic evolution in humans, with dedifferentiated tumors becoming more aggressive. Therefore, we are studying the mechanisms responsible for the phenotypic shift and for the immune suppression associated with the presence of inflammatory Amela tumors. In this model, inflammatory melanomas are associated with an enrichment in regulatory T cells and myeloid-derived suppressor cells (MDSC),17 with a TGFß and EMT-like signature,18 and with expression of arginase, pospho-STAT3 and COX2.17 We are combining vaccine approaches targeting tumor antigen P1A with strategies interfering with these immunosuppressive mechanisms.
9. Uyttenhove, C., L. Pilotte, I. Theate, V. Stroobant, D. Colau, N. Parmentier, T. Boon, and B.J. Van den Eynde. 2003. Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Nat. Med. 9:1269-1274.
10. Rohrig, U.F., S.R. Majjigapu, A. Grosdidier, S. Bron, V. Stroobant, L. Pilotte, D. Colau, P. Vogel, B.J. Van den Eynde, V. Zoete, and O. Michielin. 2012. Rational design of 4-aryl-1,2,3-triazoles for indoleamine 2,3-dioxygenase 1 inhibition. J. Med. Chem. 55:5270-5290.
11. Rohrig, U.F., L. Awad, A. Grosdidier, P. Larrieu, V. Stroobant, D. Colau, V. Cerundolo, A.J. Simpson, P. Vogel, B.J. Van den Eynde, V. Zoete, and O. Michielin. 2010. Rational design of indoleamine 2,3-dioxygenase inhibitors. J. Med. Chem. 53:1172-1189.
12. Dolusic, E., P. Larrieu, S. Blanc, F. Sapunaric, J. Pouyez, L. Moineaux, D. Colette, V. Stroobant, L. Pilotte, D. Colau, T. Ferain, G. Fraser, M. Galleni, J.M. Frere, B. Masereel, B. Van den Eynde, J. Wouters, and R. Frederick. 2011. Discovery and preliminary SARs of keto-indoles as novel indoleamine 2,3-dioxygenase (IDO) inhibitors. Eur. J. Med. Chem. 46:3058-3065.
13. Dolusic, E., P. Larrieu, S. Blanc, F. Sapunaric, B. Norberg, L. Moineaux, D. Colette, V. Stroobant, L. Pilotte, D. Colau, T. Ferain, G. Fraser, M. Galleni, J.M. Frere, B. Masereel, B. Van den Eynde, J. Wouters, and R. Frederick. 2011. Indol-2-yl ethanones as novel indoleamine 2,3-dioxygenase (IDO) inhibitors. Bioorg. Med. Chem. 19:1550-1561.
14. Pilotte, L., P. Larrieu, V. Stroobant, D. Colau, E. Dolusic, R. Frederick, E. De Plaen, C. Uyttenhove, J. Wouters, B. Masereel, and B.J. Van den Eynde. 2012. Reversal of tumoral immune resistance by inhibition of tryptophan 2,3-dioxygenase. Proc. Natl. Acad. Sci. U.S.A. 109:2497-2502.
15. Dolusic, E., P. Larrieu, L. Moineaux, V. Stroobant, L. Pilotte, D. Colau, L. Pochet, B. Van den Eynde, B. Masereel, J. Wouters, and R. Frederick. 2011. Tryptophan 2,3-Dioxygenase (TDO) Inhibitors. 3-(2-(Pyridyl)ethenyl)indoles as Potential Anticancer Immunomodulators. J. Med. Chem. 54:5320-5334.
16. Huijbers, I.J., P. Krimpenfort, P. Chomez, M.A. van der Valk, J.Y. Song, E.M. Inderberg-Suso, A.M. Schmitt-Verhulst, A. Berns, and B.J. Van den Eynde. 2006. An inducible mouse model of melanoma expressing a defined tumor antigen. Cancer Res. 66:3278-3286.
17. Soudja, S.M., M. Wehbe, A. Mas, L. Chasson, C.P. de Tenbossche, I. Huijbers, B. Van den Eynde, and A.M. Schmitt-Verhulst. 2010. Tumor-initiated inflammation overrides protective adaptive immunity in an induced melanoma model in mice. Cancer Res. 70:3515-3525.
18. Wehbe, M., S.M. Soudja, A. Mas, L. Chasson, R. Guinamard, C.P. de Tenbossche, G. Verdeil, B. Van den Eynde, and A.M. Schmitt-Verhulst. 2012. Epithelial-mesenchymal-transition-like and TGFbeta pathways associated with autochthonous inflammatory melanoma development in mice. PLoS One 7:e49419.
For a complete list of Benoît J. Van den Eynde's publications, click here.
Pilotte L, Larrieu P, Stroobant V, Colau D, Dolušić E, Frédérick R, De Plaen E, Uyttenhove C, Wouters J, Masereel B, Van den Eynde BJ. Reversal of tumoral immune resistance by inhibition of tryptophan 2,3-dioxygenase. Proc Natl Acad Sci U S A. 2012 Feb 14;109(7):2497-502.
Guillaume B, Stroobant V, Bousquet-Dubouch M-P, Colau D, Chapiro J, Parmentier N, Dalet A, Van den Eynde BJ. Analysis of the processing of seven human tumor antigens by intermediate proteasomes. J Immunol. 2012 Oct 1;189(7):3538-47. Epub 2012 Aug 27.
Huijbers IJ, Soudja SM, Uyttenhove C, Buferne M, Inderberg-Suso EM, Colau D, Pilotte L, Powis de Tenbossche CG, Chomez P, Brasseur F, Schmitt-Verhulst AM, Van den Eynde BJ. Minimal tolerance to a tumor antigen encoded by a cancer-germline gene. J Immunol. 2012 Jan 1;188(1):111-21. Epub 2011 Dec 2.
Dalet A, Robbins PF, Stroobant V, Vigneron N, Li YF, El-Gamil M, Hanada K-i, Yang JC, Rosenberg SA, Van den Eynde BJ. An antigenic peptide produced by reverse splicing and double asparagine deamidation. Proc Natl Acad Sci U S A. 2011 Jul 19;108(29):E323-31. Epub 2011 Jun 13.
Parmentier N, Stroobant V, Colau D, de Diesbach P, Morel S, Chapiro J, van Endert P, Van den Eynde BJ. Production of an antigenic peptide by insulin-degrading enzyme. Nat Immunol. 2010 May;11(5):449-54. Epub 2010 Apr 4.

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