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Abstract Neoadjuvant immune checkpoint blockade (ICB) has shown unprecedented activity in mismatch repair deficient (MMRd) colorectal cancers, but its effectiveness in MMRd endometrial cancer (EC) remains unknown. In this investigator-driven, phase I, feasibility study (NCT04262089), 10 women with MMRd EC of any grade, planned for primary surgery, received two cycles of neoadjuvant pembrolizumab (200 mg IV) every three weeks. A pathologic response (primary objective) was observed in 5/10 patients, with 2 patients showing a major pathologic response. No patient achieved a complete pathologic response. A partial radiologic response (secondary objective) was observed in 3/10 patients, 5/10 patients had stable disease and 2/10 patients were non-evaluable on magnetic resonance imaging. All patients completed treatment without severe toxicity (exploratory objective). At median duration of follow-up of 22.5 months, two non-responders experienced disease recurrence. In-depth analysis of the loco-regional and systemic immune response (predefined exploratory objective) showed that monoclonal T cell expansion significantly correlated with treatment response. Tumour-draining lymph nodes displayed clonal overlap with intra-tumoural T cell expansion. All pre-specified endpoints, efficacy in terms of pathologic response as primary endpoint, radiologic response as secondary outcome and safety and tolerability as exploratory endpoint, were reached. Neoadjuvant ICB with pembrolizumab proved safe and induced pathologic, radiologic, and immunologic responses in MMRd EC, warranting further exploration of extended neoadjuvant treatment.

Background Patients with mismatch repair-deficient (MMRd) endometrial cancer (EC) can derive great benefit from immune checkpoint inhibitors (ICI). However not all responses and predictors of primary resistance are lacking.Methods We compared the immune tumor microenvironment of MMRd EC ICI-responders (Rs) and ICI non-responders (NRs), using spatial multiplexed immune profiling and unsupervised hierarchical clustering analysis.Results Overall, NRs exhibited drastically lower CD8+, absent terminally differentiated T cells, lack of mature tertiary lymphoid structures and dendritic cells, as well as loss of human leukocyte antigen class I. However, no single marker could predict R versus NR with confidence. Clustering analysis identified a combination of four immune features that demonstrated that accurately predicted ICI response, with a discriminative power of 92%. Finally, 80% of NRs lacked programmed death-ligand 1, however, 60% exhibited another actionable immune checkpoint (T-cell immunoglobulin and mucin containing protein-3, indoleamine 2,3-dioxygenase 1, or lymphocyte activation gene 3).Conclusions These findings underscore the potential of immune tumor microenvironment features for identifying patients with MMRd EC and primary resistance to ICI who should be oriented towards trials testing novel immunotherapeutic combinations.

Abstract Background CD103 is an integrin specifically expressed on the surface of cancer-reactive T cells. The number of CD103+ T cells significantly increases during successful immunotherapy and might therefore be an attractive biomarker for noninvasive PET imaging of immunotherapy response. Since the long half-life of antibodies preclude repeat imaging of CD103+ T cell dynamics early in therapy, we therefore here explored PET imaging with CD103 Fab fragments radiolabeled with a longer (89Zr) and shorter-lived radionuclide (68Ga). Methods Antihuman CD103 Fab fragment Fab01A was radiolabeled with 89Zr or 68Ga, generating [89Zr]Zr-hCD103.Fab01A and [68Ga]Ga-hCD103.Fab01A, respectively. In vivo evaluation of these tracers was performed in male nude mice (BALB/cOlaHsd-Foxn1nu) with established CD103-expressing CHO (CHO.CD103) or CHO-wildtype (CHO.K1) xenografts, followed by serial PET imaging and ex vivo bio-distribution. Results [89Zr]Zr-hCD103.Fab01A showed high tracer uptake in CD103+ xenografts as early as 3 h post-injection. However, the background signal remained high in the 3- and 6-h scans. The background was relatively low at 24 h after injection with sufficient tumor uptake. [68Ga]Ga-hCD103.Fab01Ashowed acceptable uptake and signal-to-noise ratio in CD103+ xenografts after 3 h, which decreased at subsequent time points. Conclusion [89Zr]Zr-hCD103.Fab01A demonstrated a relatively low background and high xenograft uptake in scans as early as 6 h post-injection and could be explored for repeat imaging during immunotherapy in clinical trials. 18F or 64Cu could be explored as alternative to 68Ga in optimizing half-life and radiation burden of the tracer.

Purpose CD103, an integrin specifically expressed on the surface of cancer-reactive T cells, is significantly increased during successful immunotherapy across human malignancies. In this study, we describe the generation and zirconium-89 (89Zr) radiolabeling of monoclonal antibody (mAb) clones that specifically recognize human CD103 for non-invasive immune positron-emission tomography (PET) imaging of T cell infiltration as potential biomarker for effective anticancer immune responses.Experimental design First, to determine the feasibility of anti-CD103 immuno-PET to visualize CD103-positive cells at physiologically and clinically relevant target densities, we developed an 89Zr-anti-murine CD103 PET tracer. Healthy, non-tumor bearing C57BL/6 mice underwent serial PET imaging after intravenous injection, followed by ex vivo biodistribution. Tracer specificity and macroscopic tissue distribution were studied using autoradiography combined with CD103 immunohistochemistry. Next, we generated and screened six unique mAbs that specifically target human CD103 positive cells. Optimal candidates were selected for 89Zr-anti-human CD103 PET development. Nude mice (BALB/cOlaHsd-Foxn1nu) with established CD103 expressing Chinese hamster ovary (CHO) or CHO wild-type xenografts were injected with 89Zr-anti-human CD103 mAbs and underwent serial PET imaging, followed by ex vivo biodistribution.Results 89Zr-anti-murine CD103 PET imaging identified CD103-positive tissues at clinically relevant target densities. For human anti-human CD103 PET development two clones were selected based on strong binding to the CD103+ CD8+ T cell subpopulation in ovarian cancer tumor digests, non-overlapping binding epitopes and differential CD103 blocking properties. In vivo, both 89Zr-anti-human CD103 tracers showed high target-to-background ratios, high target site selectivity and a high sensitivity in human CD103 positive xenografts.Conclusion CD103 immuno-PET tracers visualize CD103 T cells at relevant densities and are suitable for future non-invasive assessment of cancer reactive T cell infiltration.

Reactivation of tumor infiltrating T lymphocytes (TILs) with immune checkpoint inhibitors or co-stimulators has proven to be an effective anti-cancer strategy for a broad range of malignancies. However, epithelial ovarian cancer (EOC) remains largely refractory to current T cell-targeting immunotherapeutics. Therefore, identification of novel immune checkpoint targets and biomarkers with prognostic value for EOC is warranted. Combining multicolor immunofluorescent staining’s with single cell RNA-sequencing analysis, we here identified a TIM-3/CXCL13-positive tissue-resident memory (CD8/CD103-positive) T cell (Trm) population in EOC. Analysis of a cohort of ~175 patients with high-grade serous EOC revealed TIM-3-positive Trm were significantly associated with improved patient survival. As CXCL13-positive CD8-positive T cells have been strongly linked to patient response to anti-PD1 immune checkpoint blockade, combinatorial TIM-3 and PD-1 blockade therapy may be of interest for the (re)activation of anti-cancer immunity in EOC.

Activation of STimulator of INterferon Genes (STING) is important for induction of anti-tumor immunity. A dysfunctional STING pathway is observed in multiple cancer types and associates with poor prognosis and inferior response to immunotherapy. However, the association between STING and prognosis in virally induced cancers such as HPV-positive cervical cancer remains unknown. Here, we investigated the prognostic value of STING protein levels in cervical cancer using tumor tissue microarrays of two patient groups, primarily treated with surgery (n = 251) or radio(chemo)therapy (n = 255). We also studied CD103, an integrin that marks tumor-reactive cytotoxic T cells that reside in tumor epithelium and that is reported to associate with improved prognosis. Notably, we found that a high level of STING protein was an independent prognostic factor for improved survival in both the surgery and radio(chemo)therapy group. High infiltration of CD103+ T cells was associated with improved survival in the radio(chemo)therapy group. The combination of STING levels and CD103+ T cell infiltration is strongly associated with improved prognosis. We conclude that combining the prognostic values of STING and CD103 may improve the risk stratification of cervical cancer patients, independent from established clinical prognostic parameters.

Epithelial Ovarian cancer (EOC) is the most lethal gynecological malignancy and has limited curative therapeutic options. Immunotherapy for EOC is promising, but clinical efficacy remains restricted to a small percentage of patients. Several lines of evidence suggest that the low response rate might be improved by combining immunotherapy with carboplatin and paclitaxel, the standard-of-care chemotherapy for EOC. Here, we assessed the immune contexture of EOC tumors, draining lymph nodes, and peripheral blood mononuclear cells during carboplatin/paclitaxel chemotherapy. We observed that the immune contexture of EOC patients is defined by the tissue of origin, independent of exposure to chemotherapy. Summarized, draining lymph nodes were characterized by a quiescent microenvironment composed of mostly non-proliferating naïve CD4 + T cells. Circulating T cells shared phenotypic features of both lymph nodes and tumor-infiltrating immune cells. Immunologically ‘hot’ ovarian tumors were characterized by ICOS, GITR, and PD-1 expression on CD4 + and CD8 + cells, independent of chemotherapy. The presence of PD-1 + cells in tumors prior to, but not after, chemotherapy was associated with disease-specific survival (DSS). Accordingly, we observed high MHC-I expression in tumors prior to chemotherapy, but minimal MHC-I expression in tumors after neoadjuvant chemotherapy, even though there were no differences in the number of tumor-infiltrating lymphocytes (TIL) in both groups. We therefore speculate that the TIL influx into the chemotherapy tumor microenvironment may be a consequence of the general inflammatory nature of chemotherapy-experienced tumors. Strategies to upregulate MHC-I during or after neoadjuvant chemotherapy may thus improve treatment outcome in these patients.

Human papilloma virus (HPV)-induced cervical cancer constitutively expresses viral E6/E7 oncoproteins and is an excellent target for T cell-based immunotherapy. However, not all tumor-infiltrating T cells confer equal benefit to patients, with epithelial T cells being superior to stromal T cells. To assess whether the epithelial T cell biomarker CD103 could specifically discriminate the beneficial antitumor T cells, association of CD103 with clinicopathological variables and outcome was analyzed in the TCGA cervical cancer data set (n = 304) and by immunohistochemistry (IHC) in an independent cohort (n = 460). Localization of CD103+ cells in the tumor was assessed by immunofluorescence. Furthermore, use of CD103 as a response biomarker was assessed in an in vivo E6/E7+ tumor model. Our results show that CD103 gene expression was strongly correlated with cytotoxic T cell markers (e.g. CD8/GZMB/PD1) in the TCGA series. In line with this, CD103+ cells in the IHC series co-expressed CD8 and were preferentially located in cervical tumor epithelium. High CD103+ cell infiltration was strongly associated with an improved prognosis in both series, and appeared to be a better predictor of outcome than CD8. Interestingly, the prognostic benefit of CD103 in both series seemed limited to patients receiving radiotherapy. In a preclinical mouse model, HPV E6/E7-targeted therapeutic vaccination in combination with radiotherapy increased the intratumoral number of CD103+ CD8+ T cells, providing a potential mechanistic basis for our results. In conclusion, CD103 is a promising marker for rapid assessment of tumor-reactive T cell infiltration of cervical cancers and a promising response biomarker for E6/E7-targeted immunotherapy.