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Tumors are populated by a multitude of immune cell types with varied phenotypic and functional properties, which can either promote or inhibit anti-tumor responses. Appropriate localization and function of these cells within tumors is critical for protective immunity, with CD8 T cell infiltration being a biomarker of disease outcome and therapeutic efficacy. Recent multiplexed imaging approaches have revealed highly complex patterns of localization for these immune cell subsets and the generation of distinct tumor microenvironments (TMEs), which can vary among cancer types, individuals, and within individual tumors. While it is recognized that TMEs play a pivotal role in disease progression, a better understanding of their composition, organization, and heterogeneity, as well as how distinct TMEs are reshaped with immunotherapy, is necessary. Here, we performed spatial analysis using multi-parameter confocal imaging, histocytometry, and CytoMAP to study the microanatomical organization of immune cells in two widely used preclinical cancer models, the MC38 colorectal and KPC pancreatic murine tumors engineered to express human carcinoembryonic antigen (CEA). Immune responses were examined in either unperturbed tumors or after immunotherapy with a CEA T cell bispecific (CEA-TCB) surrogate antibody and anti-PD-L1 treatment. CEA-TCB mono and combination immunotherapy markedly enhanced intra-tumoral cellularity of CD8 T cells, dominantly driven by the expansion of TCF1− PD1+ effector T cells and with more minor increases in TCF1+PD1+ resource CD8 T cells. The majority of infiltrating T cells, particularly resource CD8 T cells, were colocalized with dendritic cells (DCs) or activated MHCII+ macrophages, but largely avoided the deeper tumor nest regions composed of cancer cells and non-activated macrophages. These myeloid cell – T cell aggregates were found in close proximity to tumor blood vessels, generating perivascular immune niches. This perivascular TME was present in untreated samples and markedly increased after CEA-TCB therapy, with its relative abundance positively associated with response to therapy. Together, these studies demonstrate the utility of advanced spatial analysis in cancer research by revealing that blood vessels are key organizational hubs of innate and adaptive immune cells within tumors, and suggesting the likely relevance of the perivascular immune TME in disease outcome.

Diffuse large B cell lymphomas (DLBCL) are a highly heterogeneous subtype of Non Hodgkin Lymphoma (NHL), accounting for about 25% of NHL [1]. Despite an increased progression-free survival upon therapy, 40-50% of patients develop relapse/refractory disease, therefore there remains an important medical need [2]. T cell recruiting therapies, such as the CD20xCD3 T cell bi-specific antibody CD20-TCB (RG6026 or glofitamab), represent a novel approach to target all stages of DLBCL, especially those that fail to respond to multiple lines of treatment [3, 4]. We aimed for a better understanding of the molecular features related to the mode of action (MoA) of CD20-TCB in inducing Target/T cell synapse formation and human T cell recruitment to the tumor. To directly evaluate the correlation between synapse, cytokine production and anti-tumor efficacy using CD20-TCB, we developed an innovative preclinical human DLBCL in vivo model that allowed tracking in vivo human T cell dynamics by multiphoton intravital microscopy (MP-IVM). By ex vivo and in vivo approaches, we revealed that CD20-TCB is inducing strong and stable synapses between human T cell and tumor cells, which are dependent on the dose of CD20-TCB and on LFA-1 activity but not on FAS-L. Moreover, despite CD20-TCB being a large molecule (194.342 kDa), we observed that intra-tumor CD20-TCB-mediated human T cell-tumor cell synapses occur within 1 hour upon CD20-TCB administration. These tight interactions, observed for at least 72 hours post TCB administration, result in tumor cell cytotoxicity, resident T cell proliferation and peripheral blood T cell recruitment into tumor. By blocking the IFNγ-CXCL10 axis, the recruitment of peripheral T cells was abrogated, partially affecting the efficacy of CD20-TCB treatment which rely only on resident T cell proliferation. Altogether these data reveal that CD20-TCB’s anti-tumor activity relies on a triple effect: i) fast formation of stable T cell-tumor cell synapses which induce tumor cytotoxicity and cytokine production, ii) resident T cell proliferation and iii) recruitment of fresh peripheral T cells to the tumor core to allow a positive enhancement of the anti-tumor effect.

Purpose: CD40 agonists hold great promise for cancer immunotherapy (CIT) as they enhance dendritic cell (DC) activation and concomitant tumor-specific T-cell priming. However, the broad expression of CD40 accounts for sink and side effects, hampering the efficacy of anti-CD40 antibodies. We hypothesized that these limitations can be overcome by selectively targeting CD40 agonism to the tumor. Therefore, we developed a bispecific FAP-CD40 antibody, which induces CD40 stimulation solely in presence of fibroblast activation protein α (FAP), a protease specifically expressed in the tumor stroma. Experimental Design: FAP-CD40's in vitro activity and FAP specificity were validated by antigen-presenting cell (APC) activation and T-cell priming assays. In addition, FAP-CD40 was tested in subcutaneous MC38-FAP and KPC-4662-huCEA murine tumor models. Results: FAP-CD40 triggered a potent, strictly FAP-dependent CD40 stimulation in vitro. In vivo, FAP-CD40 strongly enhanced T-cell inflammation and growth inhibition of KPC-4662-huCEA tumors. Unlike nontargeted CD40 agonists, FAP-CD40 mediated complete regression of MC38-FAP tumors, entailing long-term protection. A high dose of FAP-CD40 was indispensable for these effects. While nontargeted CD40 agonists induced substantial side effects, highly dosed FAP-CD40 was well tolerated. FAP-CD40 preferentially accumulated in the tumor, inducing predominantly intratumoral immune activation, whereas nontargeted CD40 agonists displayed strong systemic but limited intratumoral effects. Conclusions: FAP-CD40 abrogates the systemic toxicity associated with nontargeted CD40 agonists. This enables administration of high doses, essential for overcoming CD40 sink effects and inducing antitumor immunity. Consequently, FAP-targeted CD40 agonism represents a promising strategy to exploit the full potential of CD40 signaling for CIT.

Introduction Cancer Immune Therapies have shown unprecedented results in improving tumor control.1–3 However, many patients are still refractory to treatment. A deeper understanding of the mode of action of the different CITs sub-classes may help improving therapeutic approaches to reach better anti-tumor response. For this reason, we developed a multi-photon intra-vital microscopy (MP-IVM) approach to study in vivo, at single cell level, the tumor microenvironment upon treatment with CD20-targeting T-cell bispecific antibodies (TCB) [4] in a preclinical model of diffuse large B cell lymphomas (DLBCL). Methods To selectively monitor clinical lead molecules in the context of human T cell responses, we developed a skinfold chamber model [5] in last generation humanized mice [6] that allows visualization, by MP-IVM, of labelled human T cells co-injected intra-dermally with WSU-DLCL2, a human DLBCL. We have used this model to investigate T cells recruitment to tumors upon CD20-TCB therapy: by intra-venously injecting labeled T cells in mice treated with selected blocking antibodies, we were able to identify dedicated pathways induced by CD20-TCB and regulating T cell influx into the tumor bed. Furthermore, we developed a user-independent quantification platform to assess changes in the dynamics of T cell motility and time of interaction with tumor cells. Results/Discussion We have developed an experimental preclinical model that aims to reduce xenoreaction (human T cell reaction against mouse tissue) by utilizing T cells derived from humanized mice, educated within murine thymus. We demonstrate that such model is optimal to quantify human T cell dynamics in vivo. We show that CD20-TCB localizes in the tumor and acts on tumor-resident T cell motility within 1 hour post i.v. injection (defined as functional PK), causing a sharp reduction in their speed (from 4 to 2 µm/min) and an increase in tumor/T cell interaction time; those changes last up to 72h post-treatment. In addition, we prove how the initial tumor/T cell interaction mediated by CD20-TCB lead to peripheral T cells recruitment into the tumor. This mechanism is dependent on the presence of tumor-resident T cells and on IFNg-CXCL10 pathway. Inhibiting any of these two parameters resulted in reduced T cells infiltration from the periphery and reduced anti-tumor efficacy. Conclusion We developed a reliable imaging and analysis pipeline to investigate in vivo T cell dynamics and recruitment and applied it to the study of CD20-TCB treatment of DLBCL model. Our approach has shed new lights into the MoA of this new class of immune-therapeutics, demonstrating that the IFNγ-CXCL10 pathway is involved in T cell recruitment upon CD20-TCB treatment. Disclosure Information F. Cremasco: A. Employment (full or part-time); Significant; Roche Glycart AG. E. Menietti: A. Employment (full or part-time); Significant; Roche Glycart AG. D. Speziale: A. Employment (full or part-time); Significant; Roche Glycart AG. J. Sam: A. Employment (full or part-time); Significant; Roche Glycart AG. S. Sammicheli: A. Employment (full or part-time); Significant; Roche Glycart AG. S. Chen: A. Employment (full or part-time); Significant; Roche Glycart AG. M. Richard: A. Employment (full or part-time); Significant; Roche Glycart AG. C. Klein: A. Employment (full or part-time); Significant; Roche Glycart AG. P. Umana: A. Employment (full or part-time); Significant; Roche Glycart AG. M. Bacac: A. Employment (full or part-time); Significant; Roche Glycart AG. S. Colombetti: A. Employment (full or part-time); Significant; Roche Glycart AG. M. Perro: A. Employment (full or part-time); Significant; Roche Glycart AG.

CSL is a key transcriptional repressor and mediator of Notch signaling. Despite wide interest in CSL, mechanisms responsible for its own regulation are little studied. CSL down-modulation in human dermal fibroblasts (HDFs) leads to conversion into cancer associated fibroblasts (CAF), promoting keratinocyte tumors. We show here that CSL transcript levels differ among HDF strains from different individuals, with negative correlation with genes involved in DNA damage/repair. CSL expression is negatively regulated by stress/DNA damage caused by UVA, Reactive Oxygen Species (ROS), smoke extract, and doxorubicin treatment. P53, a key effector of the DNA damage response, negatively controls CSL gene transcription, through suppression of CSL promoter activity and, indirectly, by increased p21 expression. CSL was previously shown to bind p53 suppressing its activity. The present findings indicate that p53, in turn, decreases CSL expression, which can serve to enhance p53 activity in acute DNA damage response of cells.

Stromal fibroblast senescence has been linked to ageing-associated cancer risk. However, density and proliferation of cancer-associated fibroblasts (CAFs) are frequently increased. Loss or downmodulation of the Notch effector CSL (also known as RBP-Jκ) in dermal fibroblasts is sufficient for CAF activation and ensuing keratinocyte-derived tumours. We report that CSL silencing induces senescence of primary fibroblasts from dermis, oral mucosa, breast and lung. CSL functions in these cells as a direct repressor of multiple senescence- and CAF-effector genes. It also physically interacts with p53, repressing its activity. CSL is downmodulated in stromal fibroblasts of premalignant skin actinic keratosis lesions and squamous cell carcinomas, whereas p53 expression and function are downmodulated only in the latter, with paracrine FGF signalling as the probable culprit. Concomitant loss of CSL and p53 overcomes fibroblast senescence, enhances expression of CAF effectors and promotes stromal and cancer cell expansion. The findings support a CAF activation-stromal co-evolution model under convergent CSL-p53 control.

Nat. Cell Biol. 17, 1193–1204 (2015); published online 24 August 2015; corrected after print 28 August 2015 An error in the print version of this Article meant that Witold W. Kilarski's name was incorrect. This has been corrected in all online versions.