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Only a subgroup of triple-negative breast cancer (TNBC) responds to immune checkpoint inhibitors (ICI). To better understand lack of response to ICI, we analyze 681 TNBCs for spatial immune cell contextures in relation to clinical outcomes and pathways of T cell evasion. Excluded, ignored and inflamed phenotypes can be captured by a gene classifier that predicts prognosis of various cancers as well as anti-PD1 response of metastatic TNBC patients in a phase II trial. The excluded phenotype, which is associated with resistance to anti-PD1, demonstrates deposits of collagen-10, enhanced glycolysis, and activation of TGFβ/VEGF pathways; the ignored phenotype, also associated with resistance to anti-PD1, shows either high density of CD163+ myeloid cells or activation of WNT/PPARγ pathways; whereas the inflamed phenotype, which is associated with response to anti-PD1, revealed necrosis, high density of CLEC9A+ dendritic cells, high TCR clonality independent of neo-antigens, and enhanced expression of T cell co-inhibitory receptors. Only a subset of triple negative breast cancer patients respond to immunotherapy. Here, the authors analysed spatial immune contextures, which can be captured by a gene classifier, in relation to genomic alterations, mechanisms of T cell evasion and response to anti-PD1 treatment.

T cell responses are generally curtailed by suppressive mechanisms within the tumor microenvironment (TME) that prevent T cell infiltration and function. Consequently, T cell-based therapies for solid tumors have yielded limited and often non-durable clinical responses. Tumors develop a hostile TME, where tumor-associated macrophages (TAMs) that initially support T cells are converted into immune suppressive TAMs that facilitate tumor evasion from T cell control. In fact, immune suppressive TAMs represent a dominant fraction of immune cells within the TME and their presence is associated with poor prognosis and resistance to immunotherapy. Often in close contact with effector T cells, TAMs directly suppress CD8 + T cells through mechanisms involving metabolic mediators, co-signaling receptors, their ligands and/or cytokines. Here, we revisit molecular interactions behind TAM-mediated suppression of T cell responses and address the potential targeting of such molecules and pathways to re-boost anti-tumor T cell immunity. This perspective, focusing on molecular interactions between TAM and T cells, may aid the improvement of T cell-based therapies for solid tumors.

Spatial distribution of intra-tumoral immune cell populations is considered a critical determinant of tumor evolution and response to therapy. The accurate and systemic search for contexture-based predictors would be accelerated by methods that allow interactive visualization and interrogation of tumor micro-environments (TME), independent of image acquisition platforms. To this end, we have developed the TME-Analyzer, a new image analysis tool, which we have benchmarked against 2 software tools regarding densities and networks of immune effector cells using multiplexed immune-fluorescent images of triple negative breast cancer (TNBC). With the TME-Analyzer we have identified a 10-parameter classifier, predominantly featuring cellular distances, that significantly predicted overall survival, and which was validated using multiplexed ion beam time of flight images from an independent cohort. In conclusion, the TME-Analyzer enabled accurate interactive analysis of the spatial immune phenotype from different imaging platforms as well as enhanced utility and aided the discovery of contextual predictors towards the survival of TNBC patients.

The simultaneous removal of endogenous T-cell receptor α-chains and β-chains, and the orthotopic placement of an exogenous receptor in human T cells via CRISPR gene-editing, prevents the mispairing between endogenous and transgenic receptors while preserving the cells’ function.

BackgroundTriple negative breast cancer (TNBC) lacks classical targets for hormone and/or antibody therapy, and responses to immune checkpoint inhibitors are rare and generally not sustained. However, the prognostic value of infiltrating CD8 lymphocytes and proven antigenicity of TNBC argue that this disease is amenable to adoptive T cell therapy.MethodsWe applied an integrative approach to discover and validate a tumor-restricted intracellular antigen using in silico analyses and laboratory assays such as qRT-PCR and immune-histochemical stainings of large numbers of TNBC- and healthy tissues. We selected immunogenic, non-cross-reactive epitopes derived from it through in silico predictions, immunopeptidomics and in vitro assays. We identified corresponding TCRs and screened them for their specificity using positional amino acid scanning and recognition of tumor- but not healthy cells, as well as for their sensitivity through peptide titrations, killing of patient-derived 3D organoids in vitro and killing of a TNBC cell line in vivo.ResultsWe identified the target Ropporin (ROPN1), which showed neither gene- nor protein expression in healthy human tissue databases (n=1,709) and over 15 major healthy organs according to qRT-PCR and immune-histochemical (IHC) staining. Notably, this target demonstrated homogenous protein expression in >85% of TNBC patients (n=756 gene expression; n=386 IHC). Epitope predictions and immunopeptidomics using cancer cell lines and tissues enabled identification of 11 HLA-A2-binding epitopes. Epitope-specific T cells were successfully enriched from naïve T cell repertoires for 9 epitopes, which yielded more than 25 clonal TCRs. TCRs directed against 5 epitopes were functionally expressed upon gene transfer into T cells, and TCRs directed against 3 epitopes recognized endogenously processed ROPN1. One TCR (against the FLY-A epitope) demonstrated preferential pairing between the therapeutic TCR alpha and beta chains and harbored a stringent recognition motif according to positional amino acid scanning of the cognate epitope. From an efficacy perspective, this TCR mediated dose-dependent killing of patient-derived TNBC 3D organoids in vitro and a breast cancer cell line in an in vivo murine study. Importantly, in both studies, treatment with FLY-A T cells significantly outperformed the standard of care treatments cisplatin and sacituzumab govitecam.ConclusionsROPN1 has been identified as a promising target for adoptive T cell therapy for TNBC patients and a specific and active TCR has been selected as the lead candidate for clinical development.Ethics ApprovalThis study has been approved by the Medical Ethical Committee at Erasmus MC (MEC.02.953, MEC-2020–0090).

The identification of transcriptomic alterations of HER2+ ductal carcinoma in situ (DCIS) that are associated with the density of tumor-infiltrating lymphocytes (TILs) could contribute to optimizing choices regarding the potential benefit of immune therapy. We compared the gene expression profile of TIL-poor HER2+ DCIS to that of TIL-rich HER2+ DCIS. Tumor cells from 11 TIL-rich and 12 TIL-poor DCIS cases were micro-dissected for RNA isolation. The Ion AmpliSeq Transcriptome Human Gene Expression Kit was used for RNA sequencing. After normalization, a Mann–Whitney rank sum test was used to analyze differentially expressed genes between TIL-poor and TIL-rich HER2+ DCIS. Whole tissue sections were immunostained for validation of protein expression. We identified a 29-gene expression profile that differentiated TIL-rich from TIL-poor HER2+ DCIS. These genes included CCND3, DUSP10 and RAP1GAP, which were previously described in breast cancer and cancer immunity and were more highly expressed in TIL-rich DCIS. Using immunohistochemistry, we found lower protein expression in TIL-rich DCIS. This suggests regulation of protein expression at the posttranslational level. We identified a gene expression profile of HER2+ DCIS cells that was associated with the density of TILs. This classifier may guide towards more rationalized choices regarding immune-mediated therapy in HER2+ DCIS, such as targeted vaccine therapy.

In the past three decades, the detection rate of ductal carcinoma in situ of the breast has dramatically increased due to breast screening programs. As a consequence, about 20% of all breast cancer cases are detected in this early in situ stage. Some ductal carcinoma in situ cases will progress to invasive breast cancer, while other cases are likely to have an indolent biological behavior. The presence of tumor-infiltrating lymphocytes is seen as a promising prognostic and predictive marker in invasive breast cancer, mainly in HER2-positive and triple-negative subtypes. Here, we summarize the current understanding regarding immune infiltrates in invasive breast cancer and highlight recent observations regarding the presence and potential clinical significance of such immune infiltrates in patients with ductal carcinoma in situ. The presence of tumor-infiltrating lymphocytes, their numbers, composition, and potential relationship with genomic status will be discussed. Finally, we propose that a combination of genetic and immune markers may better stratify ductal carcinoma in situ subtypes with respect to tumor evolution.

Multiple myeloma (MM) is characterized by loss of anti-tumor T cell immunity. Despite moderate success of treatment with anti-PD1 antibodies, effective treatment is still challenged by poor T cell-mediated control of MM. To better enable identification of shortcomings in T-cell immunity that relate to overall survival (OS), we interrogated transcriptomic data of bone marrow samples from eight clinical trials (n = 1654) and one trial-independent patient cohort (n = 718) for multivariate analysis. Gene expression of V-domain Ig suppressor of T cell activation (VISTA) was observed to correlate to OS [hazard ratio (HR): 0.72; 95% CI: 0.61–0.83; p = 0.005]. Upon imaging the immune contexture of MM bone marrow tissues (n = 22) via multiplex in situ stainings, we demonstrated that VISTA was expressed predominantly by CD11b+ myeloid cells. The combination of abundance of VISTA+, CD11b+ cells in the tumor but not stromal tissue together with low presence of CD8+ T cells in the same tissue compartment, termed a high VISTA-associated T cell exclusion score, was significantly associated with short OS [HR: 16.6; 95% CI: 4.54–62.50; p < 0.0001]. Taken together, the prognostic value of a combined score of VISTA+, CD11b+ and CD8+ cells in the tumor compartment could potentially be utilized to guide stratification of MM patients for immune therapies.

Treatment with adoptively transferred T cells is challenged by limited longevity of therapeutic cells within tumors. To enhance the durability of anti-tumor T cell products, we have created T cell receptors (TCRs) with built-in co-stimulatory molecules. We observed that TCRs coupled to ICOS mediated exceptionally long-term responses including delay of tumor recurrence and cures in a mouse tumor model. TCR:ICOS T cells showed enhanced and antigen-specific production of inflammatory cytokines and resistance to exhaustion. Genetic ablation of ICOS-mediated activation of the PI3K-NFκB pathway neutralized the long-term anti-tumor effects. To translate TCR:ICOS to human T cells, we identified a single amino acid change in the cytosolic tail which was necessary for functional surface expression. Notably, the optimized receptor sustained performance of human T cells upon repeated stimulation across multiple antigen specificities. Collectively, we present a novel and uniformly applicable TCR:ICOS format that supports fitter T cell products for adoptive cell therapy. Newly designed co-stimulatory TCR, with extracellular TCR-V and C domains coupled to CD28 transmembrane domain, and ICOS and CD3ε intracellular domains (in short TCR:ICOS) provides: ➢durable anti-tumor response and T cell persistence in mouse model➢inflammatory T cell phenotype and resistance to T cell exhaustion➢effects via PI3K and NFκB activation➢translation to human T cells upon single amino acid mutation in TCR:ICOS tail➢extension to multiple clinically relevant TCRs while preserving prolonged T cell fitness durable anti-tumor response and T cell persistence in mouse model inflammatory T cell phenotype and resistance to T cell exhaustion effects via PI3K and NFκB activation translation to human T cells upon single amino acid mutation in TCR:ICOS tail extension to multiple clinically relevant TCRs while preserving prolonged T cell fitness

Triple-negative breast cancer (TNBC) shows an urgent need for new therapies. We discovered Ropporin-1 (ROPN1) as a target to treat TNBC with T-cells. ROPN1 showed high and homogenous expression in 90% of primary and metastatic TNBC but not in healthy tissues. HLA-A2-binding peptides were detected via immunopeptidomics and predictions and used to retrieve T-cell receptors (TCRs) from naive repertoires. Following gene introduction into T-cells and stringent selection, we retrieved a highly specific TCR directed against the epitope FLYTYIAKV that did not recognize non-cognate epitopes from alternative source proteins. Notably, this TCR mediated killing of three-dimensional tumoroids in vitro and tumor cells in vivo and outperformed standard-of-care drugs. Finally, the T-cell product expressing this TCR and manufactured using a clinical protocol fulfilled standard safety and efficacy assays. Collectively, we have identified and preclinically validated ROPN1 as a target and anti-ROPN1 TCR T-cells as a treatment for the vast majority of TNBC patients.Competing Interest StatementThis research was conducted according to the requirements of objectivity and integrity standards. DH is listed as inventor for European patent application no. P128827EP00. MK has received research support from BMS, Roche, AstraZeneca, personal fees from AstraZeneca, Daiichi Sankyo, Domain Therapeutics, Alderaan, BMS, MSD, Gilead, Roche outside the submitted work (all paid to the NKI/AVL). RD has received research support from MSD and Bayer, personal fees from Bluebird Bio, Genticel, other support from Pan Cancer T B.V. outside the submitted work (all paid to the Erasmus MC Cancer Institute) and is listed as inventor for European patent applications P130556EP00 and P128827EP00. All other authors (DK, MvB, RW, DR, KK, MT, CYL, ATJ, RF, JM, MdB, SIB, JAD, MK, EHJD, MBS, JWM, RJMA) declare no competing interests.