Tryptophan-degrading enzymes in tumoral immune resistance

Tryptophan is required for T lymphocyte effector functions. Its degradation is one of the mechanisms selected by tumors to resist immune destruction. Two enzymes, tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase 1 (IDO1), control tryptophan degradation through the kynurenine pathway. A third protein, indoleamine 2,3-dioxygenase 2 (IDO2), was identified more recently. All three enzymes were reported to be expressed in tumors, and are candidate targets for pharmacological inhibition aimed at restoring effective anti-tumoral immunity. In this review, we compare these three enzymes in terms of structure, activity, regulation and expression in healthy and cancerous tissues, in order to appreciate their relevance to tumoral immune resistance

Combining personalized neoantigen vaccination with chemotherapy and anti-PD-1 to treat NSCLC

In this issue of Cancer Cell, Awad et al. report a phase 1b clinical trial combining a personalized vaccine NEO-PV-01 with chemotherapy and anti-PD-1 pembrolizumab in first-line metastatic non-squamous NSCLC. They demonstrate that this treatment regimen was well tolerated and induced neoantigen-specific CD4+ T cell responses with effector phenotype

Uncovering molecular actors of IDO-mediated T cell dysfunction with genome-wide CRISPR/Cas9 knockout screens

Introduction: Despite tremendous progress in cancer immunotherapy, most patients fail to benefit because of poorly characterized immune resistance mechanisms. Among these, expression of the tryptophan-catabolizing enzyme Indoleamine 2,3-dioxygenase (IDO) has been found in several tumors and associated with local immune suppression, notably by inhibiting T cell functions. However, the exact molecular pathways making T cells sensitive to IDO are still unclear. We propose to exploit the power of whole-genome single-guide RNA (sgRNA) CRISPR screens to uncover novel mechanisms of IDO-mediated T cell dysfunction. Methods: Cas9-expressing transgenic mice were crossed with mice expressing a transgenic T cell receptor (TCR) specifically recognizing a tumor antigen called P1A. Primary Cas9xTCRP1A CD8+ T cells were isolated from these mice and stimulated with P1A-expressing tumor cells. Cells were then transduced with the Teichmann retroviral genome wide CRISPR knockout library. Seven days after the first stimulation, CD8+ T cells were re-stimulated in a control (Tryptophanhigh, kynureninelow) or selective (Tryptophanlow, kynureninehigh) medium, mimicking the function of IDO in vitro. After four days of screening selection, genomic DNA was extracted from remaining living cells and sequencing libraries were prepared. sgRNA representation was then assessed by next-generation sequencing. Results: In vitro CRISPR knockout screening pipeline was successfully set up and validated. This includes: (1.) Successful transduction of the CRISPR knockout library, as well as efficient gene knockout in Cas9xTCRP1A primary CD8+ T cells. (2.) Confirming the strong inhibition of T cell proliferation and survival in the selective medium as compared to the control medium, thereby validating the screening selection strategy. (3.) Optimizing genomic DNA extraction procedure, as well as sequencing library preparation. Finally, the proposed in vitro CRISPR knockout screening was successfully launched, and analysis of potential candidate genes is ongoing. Conclusions and perspectives: Our study proposes to perform genome wide CRISPR knockout screens in T cells exposed to conditions mimicking IDO activity in vitro. Enriched sgRNA at the end of the selection should reveal genes whose inhibition improved T cell survival in Tryptophanlow/kynureninehigh medium. Resulting potential candidate targets will then be validated by knocking them out in T cells and assessing T cell functions under conditions mimicking IDO-mediated immune suppression in vitro. Similar validation of top candidate genes will also be performed in vivo in a model of adoptive cell transfer of target-knockout T cells in mice bearing IDO-expressing tumors. The identified genes/pathways involved in T cell sensitivity to Tryptophan shortage/Kynurenine enrichment should reveal new relevant mechanisms of IDO-mediated immune suppression in the tumor microenvironment

Integrating Next-Generation Dendritic Cell Vaccines into the Current Cancer Immunotherapy Landscape

Cancer immunotherapy is experiencing a renaissance spearheaded by immune checkpoint inhibitors (ICIs). This has spurred interest in 'upgrading' existing immunotherapies that previously experienced only sporadic success, such as dendritic cells (DCs) vaccines. In this review, we discuss the major molecular, immunological, and clinical determinants of existing first- and second-generation DC vaccines. We also outline the future trends for next-generation DC vaccines and describe their major hallmarks and prerequisites necessary for high anticancer efficacy. In addition, using existing data we compare DC vaccines with ICIs targeting CTLA4, PD1, and PD-L1, and argue that in various contexts next-generation DC vaccines are ready to meet some challenges currently confronting ICIs, thereby raising the need to integrate DC vaccines in future combinatorial immunotherapy regimens.status: publishe

Tumour immune rejection triggered by activation of α2-adrenergic receptors

Immunotherapy based on immunecheckpoint blockade (ICB) using antibodies induces rejection of tumours and brings clinical benefit in patients with various cancer types. However, tumours often resist immune rejection. Ongoing efforts trying to increase tumour response rates are based on combinations of ICB with compounds that aim to reduce immunosuppression in the tumour microenvironment but usually have little effect when used as monotherapies. Here we show that agonists of α2-adrenergic receptors (α2-AR) have very strong anti-tumour activity when used as monotherapies in multiple immunocompetent tumour models, including ICB-resistant models, but not in immunodeficient models. We also observed marked effects in human tumour xenografts implanted in mice reconstituted with human lymphocytes. The anti-tumour effects of α2-AR agonists were reverted by α2-AR antagonists, and were absent in Adra2a-knockout (encoding α2a-AR) mice, demonstrating on-target action exerted on host cells, not tumour cells. Tumours from treated mice contained increased infiltrating T lymphocytes and reduced myeloid suppressor cells, which were more apoptotic. Single-cell RNA-sequencing analysis revealed upregulation of innate and adaptive immune response pathways in macrophages and T cells. To exert their anti-tumour effects, α2-AR agonists required CD4+ T lymphocytes, CD8+ T lymphocytes and macrophages. Reconstitution studies in Adra2a-knockout mice indicated that the agonists acted directly on macrophages, increasing their ability to stimulate T lymphocytes. Our results indicate that α2-AR agonists, some of which are available clinically, could substantially improve the clinical efficacy of cancer immunotherapy

Inhibition of Tryptophan-Dioxygenase Activity Increases the Antitumor Efficacy of Immune Checkpoint Inhibitors

Tryptophan 2,3-dioxygenase (TDO) is an enzyme that degrades tryptophan into kynurenine and thereby induces immunosuppression. Like indoleamine 2,3-dioxygenase (IDO1), TDO is considered as a relevant drug target to improve the efficacy of cancer immunotherapy. However, its role in various immunotherapy settings has not been fully characterized. Here, we described a new small-molecule inhibitor of TDO that can modulate kynurenine and tryptophan in plasma, liver, and tumor tissue upon oral administration. We showed that this compound improved the ability of anti-CTLA4 to induce rejection of CT26 tumors expressing TDO. To better characterize TDO as a therapeutic target, we used TDO-KO mice and found that anti-CTLA4 or anti-PD1 induced rejection of MC38 tumors in TDO-KO, but not in wild-type mice. As MC38 tumors did not express TDO, we related this result to the high systemic tryptophan levels in TDO-KO mice, which lack the hepatic TDO needed to contain blood tryptophan. The antitumor effectiveness of anti-PD1 was abolished in TDO-KO mice fed on a tryptophan-low diet that normalized their blood tryptophan level. MC38 tumors expressed IDO1, which could have limited the efficacy of anti-PD1 in wild-type mice and could have been overcome in TDO-KO mice due to the high levels of tryptophan. Accordingly, treatment of mice with an IDO1 inhibitor improved the efficacy of anti-PD1 in wild-type, but not in TDO-KO, mice. These results support the clinical development of TDO inhibitors to increase the efficacy of immunotherapy of TDO-expressing tumors and suggest their effectiveness even in the absence of tumoral TDO expression.See article by Hoffmann et al., p. 19.status: publishe

Characterization of T-cell Receptors Directed Against HLA-A*01-restricted and C*07-restricted Epitopes of MAGE-A3 and MAGE-A12

The ability of T cells that have been genetically engineered to express T cell receptors (TCRs) directed against tumor antigens to mediate tumor regression has been demonstrated in several clinical trials. These TCRs have primarily targeted HLA-A*0201 restricted TCRs, as approximately 50% of Caucasians, who represent the predominant population of patients who develop melanomas, expresses this HLA class I allele. These therapies could be extended to additional patients through the use of TCRs that target epitopes that are presented by additional class I alleles that are prevalent in this population such as HLA-C*07 and HLA-A*01, which are expressed by approximately 50% and 30% of patients, respectively. Therefore, two TCRs that recognize an epitope of MAGE-A12 in the context of HLA-C*07, as well as two TCRs that recognize an epitope of MAGE-A3 in the context of HLA-A*01 were isolated from tumor reactive T cell clones and cloned in a recombinant retroviral expression vector. Comparative studies indicated that one of the two MAGE-A3 reactive TCRs and one of the two MAGE-A12 reactive TCRs were superior to the additional TCRs in conferring transduced PBMC with the capacity to recognize a broad array of antigen and MHC positive target cells. These results provide support the use of these TCRs in cancer adoptive immunotherapy trials