OncoImmunology

BackgroundChimeric antigen receptor (CAR)-T cell therapies efficacy in solid tumors remains limited, largely due to the profoundly immunosuppressive tumor microenvironment (TME) which drives CAR-T cells to dysfunction and poor persistence. A comprehensive understanding of the dynamic interplay between CAR-T cells and the TME is therefore critical for the rational design of more effective CAR-T strategies for solid cancers. MethodsHere, we performed single-cell RNA sequencing of tumor samples from immunocompetent mice treated with stroma-targeting EDA-CAR-T cells, profiling CAR-T cell states and TME programs at the peak of antitumor response and during subsequent tumor progression. ResultsOur analysis revealed a marked temporal remodeling of EDA-CAR-T cells within the TME, where early antitumor efficacy is associated with concurrent expansion of cytotoxic effector CD8 CAR-T cells and activation of memory CD4 CAR-T subsets. Moreover, EDA-CAR-T cells effectively engaged the myeloid compartment, resulting in strengthened communication networks involving T cell activation. However, by tumor progression, EDA-CAR-T cells suffered a widespread transcriptional reprogramming towards dysfunction, characterized by loss of effector programs alongside induction of exhaustion and immunoregulatory pathways within the TME, including PD-L1/PD-L2 and TGF{beta} signaling, which impairs sustained immune responses. Notably, early CAR-T cell activation led to increased susceptibility to TME-mediated immunosuppression, revealing EDA-CAR-T-specific soluble galectin-mediated cell-to-cell interaction networks. ConclusionsTogether, this works offers a high-resolution view of CAR-T cell dynamics within the solid TME, uncovering cellular and molecular mechanisms of rapid functional decline and identifying regulatory pathways within the TME that can be exploited to improve CAR-T cell therapy efficacy in solid tumors. KEY MESSAGES OF THE ARTICLEO_ST_ABSWhat is already known on this topicC_ST_ABSThe determinants of CAR-T cell therapeutic efficacy in solid tumors remain poorly defined, largely due to the complexity of the immunosuppressive tumor microenvironment. In this effort, it is necessary to perform comprehensive and detailed mechanistic studies that capture CAR-T cell dynamics within the solid tumor microenvironment to understand treatment failure. What this study addsWe performed single-cell profiling of stroma-targeting EDA-CAR-T cells, revealing their dynamic reprogramming toward dysfunction within the solid tumor microenvironment. We dissected CAR-T cell states and their cell-to-cell interactions with the tumor microenvironment across response and tumor progression and identified mechanisms linking CAR-T cell functionality and therapeutic failure. How this study might affect research, practice or policyThis study provides comprehensive mechanistic insights from an immunocompetent model that can be leveraged to identify shared determinants of CAR-T cell functionality in solid tumors and potentially guide the rational development of improved CAR-T cell therapies.

Despite vaccination, cervical tumors remain a health issue and require treatment improvement. In 2023, Pembrolizumab, an anti-PD-1 immune checkpoint blockade (ICB), was introduced in the case of advanced or metastatic cervical tumors. This treatment significantly increased progression free survival from 20% to 50%. However, some patients remain resistant to anti-PD-1 treatment, which calls for new targets. In our study, we highlighted poliovirus receptor (PVR) and Nectin-2 as potential ICB targets. Indeed, PVR and Nectin-2 are TIGIT ligands, an immunomodulatory checkpoint expressed by regulatory T cells or exhausted T cells. The binding of PVR or Nectin-2 to TIGIT maintains the immunosuppressive signal in the immune cells allowing tumor progression. Furthermore, we observed that PVR and Nectin-2 were highly expressed on tumor cells and tumor associated macrophages (TAMs) accross the different histological subsets of cervical tumors. Therefore, we hypothesized that using anti-PVR and anti-Nectin-2 anti-bodies would lift immunosuppression in cervical tumors. To that end, we used CyTOF to asses precise immunophenotyping of the targets ex vivo, high throughput confocal microscopy to assess phagocytosis, monocyte derived macrophages (MDM) coupled with 3D cell culture models to assess the impact of our treatment on MDM and TAM repolarization and tumor growth. We could demonstrate that our treatment repolarized macrophages towards an inflammatory profile and that this was followed by a reactivation of macrophage cytotoxic function such as phagocytosis. We also demonstrated that anti-PVR and Nectin-2 treatment allowed the control of tumor growth in 2D and 3D cell culture models. We could also develop a pre-clinical model of autologous cell culture from cervical cancer patients. Using the MIVO technology, which combine organotypic culture and fluidics, we could assess peripheral blood mononuclear cells recruitment towards tumor cells in the presence or absence of anti-PVR and anti-Nectin-2. In conclusion we could demonstrate that targeting macrophages via the PVR/Nectin-2 couple reactivates cervical tumor growth control and improves immune cell recruitment.

PurposeTriple-negative breast cancers (TNBC) account for 15% of all breast cancers but carry the worst prognosis. Because of their heterogenicity, these tumors are not all prone to targeted therapies. However, due to their high immune infiltration, targeting their immune microenvironment is of tremendous interest and is becoming the standard of care for high-risk early-stage TNBC. Nevertheless, the characterization of this immune infiltrate is often limited to general tumor-infiltrating lymphocytes (TILs) counting, without characterization of lymphocytes subtypes. Thus, we aimed at precisely characterizing these sub-populations and evaluating their prognostic significance. MethodsWe selected 91 TNBC tumors for which we had both the TILs count on hematoxylin and eosin (H&E) slides determined by an expert pathologist and the immune microenvironment cell subtypes characterization using flow cytometry (FC). We then compared the prognostic value of immune microenvironment subpopulations vs total TILs count. ResultsTNBCs contained a mean of 22.8{+/-}25.9% TILs in the tumor surface area, including mainly CD4+ helper T lymphocytes (14.1%), mostly Th2 (11.7%), CD8+ cytotoxic T lymphocytes (11.1%), and myeloid cells (8.4%) including antigen presenting cells (APC). The TILs count was correlated with the abundance of these cellular subpopulations (p[≤]0.004). TILs percentage was predictive of overall survival (OS) in univariate analysis (p=0.044), high APC infiltration was predictive of relapse-free survival (RFS) in univariate analysis (p[≤]0.030), and Th2 infiltration was predictive of both RFS and OS in univariate (p=0.009, 0.008 respectively) and multivariate analyses (p=0.002, 0.010 respectively). ConclusionThe characterization of TILs composition is essential to better understand the potential antitumoral functions of these cells, and to strongly improve the associated prognostic and predictive values. We here demonstrate that Th2 subpopulation is associated with a better overall survival in TNBC and could be of use to predict response to the newly used immunotherapies.

Immune checkpoint blockade (ICB) has demonstrated clinical efficacy in several cancers, including melanoma, lung, colorectal, and liver malignancies. However, a substantial proportion of patients fail to respond, underscoring the need for alternative immunotherapeutic strategies capable of overcoming resistance to conventional checkpoint inhibition. One such strategy involves targeting intracellular inhibitory immune checkpoints that regulate effector lymphocyte function. Rasal1, a Ras GTPase-activating protein, has been shown to negatively regulate T cell-mediated antitumor immunity. In this study, we further characterized the impact of Rasal1 impairment on tumor progression, T cell stemness, and effector function. Using an endonuclease-mediated mutation targeting the C2 domain of Rasal1, we demonstrate that Rasal1-impaired (Rasal1i) mice exhibit significantly reduced tumor growth across multiple murine cancer models. Rasal1i mice displayed increased intratumoral CD8+ T cell accumulation, activation, cytolytic capacity, and enhanced Wnt signaling. Tumor-infiltrating lymphocytes additionally exhibited increased progenitor and stem-like memory phenotypes. Notably, Rasal1 inhibition prolonged survival and potentiated PD-1 therapy in a resistant PD-L1-expressing B16F10 melanoma model. Collectively, these findings identify Rasal1 as an intracellular inhibitory immune checkpoint that constrains T cell stemness and antitumor function, and support its further evaluation as a therapeutic target for cancer immunotherapy.

BackgroundHigh grade serous cancer (HGSC) remains a highly fatal malignancy with less than 50% of patients surviving 5 years after diagnosis. Despite its high mutational burden, HGSC is relatively refractory to checkpoint immunotherapy, suggesting that additional features of the cancer and its interactions with the immune system remain to be understood. Natural killer (NK) cells may contribute to HGSC control, but the role(s) of this population or its subsets in this disease are poorly understood. MethodsWe used a TMA containing duplicate treatment-naive tumors from 1145 patients with HGSC and a custom staining panel to simultaneously measure macrophages, T cells and NK cells, separating NK cells based on CD16a expression. Using pathologist-validated digital pathology, machine learning, computational analysis and Pearsons correlations, we quantitated infiltrating immune cell density, co-infiltration and co-localization with spatial resolution to tumor region. We compared the prognostic value of innate, general, and adaptive immune cell "neighborhoods" to define characteristics of HGSC tumors predictive for progression-free survival and used flow cytometry to define additional features of the CD16adim NK cell subset. ResultsNK cells were observed in >95% of tumor cores. Intrastromal localization of CD16alow and CD16ahigh NK cells was associated with shorter and longer progression-free survival, respectively. CD16ahigh NK cells most frequently co-localized with T cells and macrophages; their proximity was termed an "adaptive" neighborhood. We find that tumors with more area represented by adaptive immune cell neighborhoods corresponded to superior progression free survival. In contrast, CD16alow NK cells did not co-infiltrate with other immune cell types, and expressed the ectonucleotidases, CD39 and CD73, which have been previously associated with poor prognosis in patients with HGSC. ConclusionsProgression-free survival for patients with HGSC may be predicted by the subset of NK cells within the tumor infiltrate (i.e. CD16ahigh vs. CD16alow). NK cell subtypes were associated predictable co-infiltrating and co-localizing leukocyte subsets, suggesting that their presence and activity may influence, or be influenced by the tumor microenvironment. Our data suggest that immunotherapeutic strategies for HGSC should consider the constitution of NK cell subsets and may benefit from mobilizing and activating CD16high NK cells.

Two hypermutated colon cancer cases with patient-derived cell lines, peripheral and tumor-infiltrating T cells available were selected for detailed investigation of immunological response. T cells co-cultured with autologous tumor cells showed only low levels of pro-inflammatory cytokines and failed at tumor recognition. Similarly, treatment of co-cultures with immune checkpoint inhibitors (ICI) did not boost antitumor immune responses. Since proteinase inhibitor 9 (PI-9) was detected in tumor cells, a specific inhibitor (PI-9i) was used in addition to ICI in T cell cytotoxicity testing. However, only pre-stimulation with tumor-specific peptides (cryptic and neoantigenic) significantly increased recognition and elimination of tumor cells by T cells independently of ICI or PI-9i. We showed, that ICI resistant tumor cells can be targeted by tumor-primed T cells and also demonstrated the superiority of tumor-naive peripheral blood T cells compared to highly exhausted tumor-infiltrating T cells. Future precision immunotherapeutic approaches should include multimodal strategies to successfully induce durable anti-tumor immune responses.

Immune checkpoint blockade (ICB) is the standard of care for recurrent/metastatic head and neck squamous cell carcinoma (HNSCC), yet efficacy remains low. The current approach for predicting the likelihood of response to ICB is a single proportional biomarker (PD-L1) expressed in immune and tumor cells (Combined Positive Score, CPS) without differentiation by cell type, potentially explaining its limited predictive value. Tertiary Lymphoid Structures (TLS) have shown a stronger association with ICB response than PD-L1. However, their exact composition, size, and spatial biology in HNSCC remain understudied. A detailed understanding of TLS is required for future use as a clinically applicable predictive biomarker. MethodsPre-ICB tumor tissue sections were obtained from 9 responders (complete response, partial response, or stable disease) and 11 non-responders (progressive disease) classified via RECISTv1.1. A custom multi-immunofluorescence (mIF) staining assay was designed, optimized, and applied to characterize tumor cells (pan-cytokeratin), T cells (CD4, CD8), B cells (CD19, CD20), myeloid cells (CD16, CD56, CD163), dendritic cells (LAMP3), fibroblasts ( Smooth Muscle Actin), proliferative status (Ki67) and immunoregulatory molecules (PD1). Spatial metrics were compared among groups. Serial tissue sections were scored for TLS in both H&E and mIF slides. A machine learning model was employed to measure the effect of these metrics on achieving a response to ICB (SD, PR, or CR). ResultsA higher density of B lymphocytes (CD20+) was found in responders compared to non-responders to ICB (p=0.022). A positive correlation was observed between mIF and pathologist identification of TLS (R2= 0.66, p-value= <0.0001). TLS trended toward being more prevalent in responders to ICB (p=0.0906). The presence of TLS within 100 {micro}m of the tumor was associated with improved overall (p=0.04) and progression-free survival (p=0.03). A multivariate machine learning model identified TLS density as a leading predictor of response to ICB with 80% accuracy. ConclusionImmune cell densities and TLS spatial location within the tumor microenvironment play a critical role in the immune response to HNSCC and may potentially outperform CPS as a predictor of ICB response.

BackgroundLung cancer is the leading cause of cancer-related deaths worldwide, with brain metastasis (BM) occurring in 40% of advanced non-small cell lung cancer (NSCLC) patients. In 15% of these patients, the brain is the only affected organ (oligo-metastasis), corresponding to improved prognosis compared to widespread disease. Thus far, it is unknown if the metastatic dissemination to the brain without systemic metastases is a consequence of the immune systems ability to control systemic tumor outgrowth. MethodsHere, we investigated the local and peripheral immune cell composition in NSCLC BM patients, and identified new immune patterns related to the occurrence of brain metastases either as oligo- or poly-metastatic disease. ResultsThe multi-parametric immune phenotyping of peripheral blood revealed a downregulation of KLRG1 in CD8+ T-cells and an increase in CD4+ TH17 cells and elevated IL-17 levels in the blood of all NSCLC BM patients compared to healthy individuals. In addition, BM patients CD4+ T cells showed less CD73 expression with reduced effector memory differentiation. Furthermore, we observed less intra-tumoral infiltration in tumor tissues and a distinctive CD4+ T-cell profile in oligo-synchronous BM, both in the tumor microenvironment and peripheral blood compared to poly-metastatic BM patients. Moreover, 5'-ectonucleotidase CD73 was significantly upregulated in CD4 and T-regulatory cells of oligo-synchronous BM. ConclusionsThese results indicate that oligo-synchronous BM exhibits a more pronounced alteration in the CD4 T-cell immune profile both locally at the tumor site and systemically. Key PointsO_LIBM patients exhibit a skewed systemic immune profile, characterized by downregulation of KLRG1 in CD8+ and induction of TH17/IL-17 axis and CD73 in CD4+ T-cells. C_LIO_LIOligo-synchronous BM displayed a distinct CD4+ T-cell profile in both TME and peripheral blood. C_LI Importance of the StudyThis study presents a novel insight into immune profiles of brain metastasis types in NSCLC patients. Examining tissues and PBMCs sheds light on the disease and uncovers unique immune responses within distinct brain metastasis patterns. This research offers valuable knowledge for improved understanding and identifying potential prognosis markers.

BackgroundIn most patients with relapsed or metastatic head and neck squamous cell carcinoma (rmHNSCC), immunotherapy with PD-1 targeting antibodies does not yield durable responses. PD-L1 tissue expression - the most commonly assessed marker for checkpoint inhibiting antibodies - is an insufficient predictor of treatment outcome. MethodsWe evaluated various blood and tissue-based biomarkers in the context of immune checkpoint blockade-based treatment to find suitable response biomarkers in a clinical trial cohort of patients with rmHNSCC. ResultsThe PD-L1 expression level in tumor or tumor microenvironment was not associated with treatment benefit. In contrast, inflammation-related markers such as IL-6, high peripheral neutrophils and high levels of cell-free DNA, as well as markers related to adaptive immune dysfunction such as altered T cell dynamics and secretion of immune checkpoint molecules, were associated with poor clinical outcomes. Patients lacking these high-risk markers performed remarkably well on inhibition of immune checkpoints with pembrolizumab. ConclusionsBiomarker-guided patient selection for pembrolizumab monotherapy or novel combinatorial approaches - potentially including anti-inflammatory agents - for patients with immune-impaired, inflammatory profiles may be the next step in personalizing immunotherapy for these hard-to-treat patients.

Bladder cancer (BC) is a prevalent malignancy with poor prognosis in advanced stages. While immune checkpoint blockade has revolutionized immunotherapy, its efficacy remains limited for most advanced BC patients. The detailed characterization of BCs tumor microenvironment (TME) is a prerequisite to understand these mechanisms of resistance and to develop new therapeutic strategies. In this study, we used a genetically engineered BC mouse model resistant to anti-PD1 treatment, and BC patient samples, to investigate the evolution of tumor-associated macrophages (TAMs) during BC progression. We identified a subset of pro-tumor TAMs expressing CXCR4, predominantly found in advanced stages of BC-bearing mice and in half of muscle-invasive BC patients from the studied cohort. Interestingly, CXCR4+ TAM-rich regions were associated with CD8 T cell-excluded areas in both mice and patients. Administration of a small molecule CXCR4 inhibitor significantly reduced the number of pro-tumor TAMs within the tumor and markedly prolonged mouse survival. Incorporating this inhibitor into a tri-immunotherapy regimen further enhanced survival, highlighting the potential of targeting multiple pathways to strongly enhance anti-tumor effects and offering new hope for improving immunotherapy in advanced BC.

Immunosuppressive tumour microenvironments (TME) reduce the effectiveness of immune responses in cancer. Non-haematopoietic mesenchymal stromal cells (MSC), the precursor to cancer associated fibroblasts (CAFs), dictate tumour progression by enhancing immune cell suppression. Hyper-sialylation of glycans promotes immune evasion in cancer, but the role of sialyation in stromal cell-mediated immunosuppression is unknown. Here we study changes in sialyltransferase (ST) enzymes and associated surface expressed sialic acid in stromal cells following inflammatory and tumour secretome conditioning. We show that tumour conditioned stromal cells have increased levels of sialyltransferases, 2,3/6 linked sialic acid and siglec ligands. In tumour models of solid (colorectal cancer) and haematological (multiple myeloma) stromal rich tumours, stromal cell sialylation is associated with enhanced immunosuppression. Using datasets and patient samples, we confirm that targeting sialylation in tumour stromal cells reverses immune cell exhaustion. Targeting stromal cell sialylation may represent a novel immune checkpoint to reactivate anti-tumour immunity.

Metastatic renal cell carcinoma (RCC) has a poor prognosis. Recent advances have shown beneficial responses to immune checkpoint inhibitors, such as anti-PD-1 or anti-PD-L1 antibodies. As only a subset of RCC patients respond, alternative strategies should be explored. Patients refractory to anti-PD-1 therapy may benefit from autologous tumor infiltrating lymphocyte (TIL) therapy. Even though efficient TIL expansion was reported from RCC lesions, it is not well established how many RCC TIL products are tumor-reactive, how well they produce pro-inflammatory cytokines in response to autologous tumors, and whether their response correlates with the presence of specific immune cells in the tumor lesions. We here compared the immune infiltrate composition of RCC lesions with that of autologous kidney tissue of 18 RCC patients. T cell infiltrates were increased in the tumor lesions, and CD8+ T cell infiltrates were primarily of effector memory phenotype. Nine out of 16 (56%) tested TIL products we generated were tumor-reactive, as defined by CD137 upregulation after exposure to autologous tumor digest. Tumor reactivity was found in particular in TIL products originating from tumors with a high percentage of infiltrated T cells compared to autologous kidney, and coincided with increased ex vivo CD25 expression on CD8+ T cells. Importantly, although TIL products had the capacity to produce the key effector cytokines IFN-{gamma}, TNF- or IL-2, they failed to do so in response to autologous tumor digests. In conclusion, TIL products from RCC lesions contain tumor-reactive T cells. Their lack of tumor-specific cytokine production requires further investigation of immunosuppressive factors in RCC and subsequent optimization of RCC-derived TIL culture conditions.

BackgroundTNFRF-14/HVEM is the ligand for BTLA and CD160 negative immune co-signaling molecules as well as viral proteins. Its expression is dysregulated with an overexpression in tumors and a connection with tumors of adverse prognosis. MethodsWe developed C57BL/6 mouse models co-expressing human huBTLA and huHVEM as well as antagonistic monoclonal antibodies (mAbs) that completely prevent the interactions of HVEM with its ligands. ResultsHere, we show that the anti-HVEM18-10 mAb increases primary human {beta}-T cells activity alone (CIS-activity) or in the presence of HVEM-expressing lung or colorectal cancer cells in vitro (TRANS-activity). Anti-HVEM18-10 synergizes with anti-PD-L1 mAb to activate T cells in the presence of PDL-1 positive tumors, but is sufficient to trigger T cell activation in the presence of PD-L1 negative cells. In order to better understand HVEM18-10 effect in vivo and especially disentangle its CIS and TRANS effects, we developed a knock-in (KI) mouse model expressing human BTLA (huBTLA+/+) and a KI mouse model expressing both human BTLA and human HVEM (huBTLA+/+ /huHVEM+/+ (DKI)). In vivo pre-clinical experiments performed in both mouse models showed that HVEM18-10 treatment was efficient to decrease human HVEM+ tumor growth. In the DKI model, anti-HVEM 18-10 treatment induces a decrease of exhausted CD8+ T cells and regulatory T cells and an increase of Effector memory CD4+ T cells within the tumor. Interestingly, mice which completely rejected tumors ({+/-} 20%) did not develop tumors upon re-challenge in both settings, therefore showing a marked T cell-memory phenotype effect. ConclusionsAltogether, our preclinical models validate anti-HVEM18-10 as a promising therapeutic antibody to use in clinics as a monotherapy or in combination with existing immunotherapies (anti-PD1/anti-PDL-1/anti-CTLA-4).

BackgroundProfessional antigen-presenting dendritic cells (DC) are critical for anti-tumor immune responses, yet patients with glioblastoma, an aggressive primary brain tumor that responds poorly to current investigational immunotherapies, appear to be deficient in DC. The extent of this deficiency, the specific DC subsets affected, and the causative mechanisms remain undefined. Furthermore, DCs in other brain tumors have not been systematically investigated. MethodsHigh-parameter flow cytometry was used to profile circulating and intra-tumoral DCs in patients with glioblastoma, low-grade gliomas, and brain metastases, and non-CNS cancers. Plasma DC growth factors were quantified using ELISA. We also evaluated single-cell RNA sequencing (scRNAseq) datasets to compare intra-tumoral DCs in brain and lung tumors, and quantified DC number and phenotype in three intracranial mouse brain tumor models. ResultsOur studies reveal a profound systemic reduction of multiple DC subsets in the blood of patients with diverse brain tumors, coupled with reduced DC activation marker expression and lower plasma levels of FLT3L and G-CSF. Furthermore, scRNAseq analyses revealed reduced intra-tumoral DCs in glioblastoma compared to lung tumors. Circulating DC numbers inversely correlated with perioperative corticosteroid dose in patients with or without a brain tumor. However, brain tumor patients not receiving corticosteroids also had reduced DCs, suggesting a direct effect of the brain tumor. This was supported by our observation of systemic DC defects in mouse brain tumor models. ConclusionsWe reveal profound DC defects in patients with brain tumors, which may contribute to current difficulties in developing effective immunotherapies for glioblastoma. SummaryWe demonstrate multiple DC defects in patients with brain tumors. This includes a profound reduction in circulating DC number, diminished activation marker expression and growth factor levels in cancer patients with brain tumors compared to those without, and reduced intra-tumoral DCs in brain compared to lung tumors. This is the first time DC subsets have been fully characterized in a range of brain tumor patients. We show that corticosteroid usage is closely associated with DC defects, highlighting the adverse effects of a standard symptomatic treatment on these critical immune cells. However, tumors located within the brain also directly contribute to DC defects. We identified several mouse brain tumor models that can be used to further the understanding of this endogenous DC deficiency and to develop approaches to restore DCs, ultimately leading to new combination immunotherapies for the treatment of brain cancers. HighlightsO_LIDCs are reduced in brain tumor patients and mice with intracranial tumors. C_LIO_LIDCs are rare within glioblastoma tumor tissue. C_LIO_LIThe presence of a brain tumor and corticosteroid use are both associated with DC defects. C_LI

BackgroundImmune checkpoint blockade (ICB) has been approved as first-line or second-line therapies for an expanding list of malignancies. T cells recognizing mutation-derived neoantigens are hypothesized to play a major role in tumor elimination. However, the dynamics and characteristics of such neoantigen-reactive T cells (NARTs) in the context of ICB are still limitedly understood. MethodsTo explore this, tumor biopsies and peripheral blood were obtained pre- and post-treatment from 20 patients with solid metastatic tumors, in a Phase I basket trial. From whole-exome sequencing and RNA-seq data, patient-specific libraries of neopeptides were predicted and screened with DNA barcode-labeled MHC multimers for CD8+ T cell reactivity, in conjunction with the evaluation of T cell phenotype. ResultsWe were able to detect NARTs in the peripheral blood and tumor biopsies for the majority of the patients; however, we did not observe any significant difference between the disease control and progressive disease patient groups, in terms of the breadth and magnitude of the detected NARTs. We also observed that the hydrophobicity of the peptide played a role in defining neopeptides resulting in NARTs response. A trend towards a treatment-induced phenotype signature was observed in the NARTs post-treatment, with the appearance of Ki67+ CD27+ PD-1+ subsets in the PBMCs and CD39+ Ki67+ TCF-1+ subsets in the TILs. Finally, the estimation of T cells from RNAseq was increasing post versus pre-treatment for disease control patients. ConclusionOur data demonstrates the possibility of monitoring the characteristics of NARTs from tumor biopsies and peripheral blood, and that such characteristics could potentially be incorporated with other immune predictors to understand further the complexity governing clinical success for ICB therapy.

Fc{gamma} RIIA (CD32A) and their ligands, including the immunoglobulin Fc fragment and pentraxins, are key players in a variety of innate immune responses. Still unclear is whether additional ligands of CD32A do exist. The objective of this study is to demonstrate that CD32A-chimeric receptor (CR) can be utilized for the identification of CD32A cell surface ligand(s). Among fifteen cancer cell lines tested, CD32A-CR T cells recognized three of breast cancer (BC) including the MDA-MB-468 and one colorectal carcinoma (HT29) in the absence of targeting antibodies. Conjugation of sensitive BC cells with CD32A-CR T cells induced CD32A polarization and down-regulation, CD107 release, and mutual cell elimination in vitro. Conversely, normal fibroblasts and myoblasts were not affected while normal HUVEC cells promoted CD32A down-regulation. CD32A-CR T cell activity was not inhibited by human IgGs or human serum, but; it was rather enhanced by cetuximab antibody. RNAseq analysis of sensitive vs resistant BC cells identified a fingerprint of 42 genes predicting the sensitivity of BC cells to CD32A-CR T cells and their association with favorable prognostic significance in advanced BC patients. Our data also identify ICAM 1 as a major regulator of CD32A-CR T cell-mediated cytotoxicity. Finally, CD32A-CR T cell administration protected immunodeficient mice from subcutaneous growth of MDA-MB-468 cells in the absence of tumor-specific antibodies. These data indicate that CD32A-CR can be utilized for the identification of (1) cell surface CD32A ligand(s); (2) rational therapeutic strategies to target BC; and (3) novel transcriptomic signatures prognostically relevant for advanced BC patients.

Chimeric antigen receptor T (CAR T) cells represent a promising therapeutic option for a variety of cancers, including solid tumors. Nectin-4 is a cell adhesion molecule expressed at different levels in many solid tumors, including breast and urothelial carcinoma (UC). Enfortumab vedotin (EV), an antibody-drug conjugate (ADC) against Nectin-4, has significantly improved survival in patients with metastatic UC. Skin adverse reactions are frequently observed due to Nectin-4 expression in the epidermis. Here, we developed second-generation CAR T cells against Nectin-4 (N4CART) with a scFv that does not recognize human Nectin-4 expressed in skin keratinocytes. To study the effects of N4CART cell therapy, we used preclinical models of cell-derived xenografts (CDX) and patient-derived xenograft (PDX) of breast cancer expressing moderate to high levels of Nectin-4. We showed a marked efficacy with induction of remissions. Interestingly, N4CART cells kill Nectin-4-positive breast, urothelial and colon tumor cells, which are resistant to EV. Thus, N4CART cells represent a valuable and safe therapy for the treatment of patients with Nectin-4 expressing tumors, including those that are resistant to EV. Finally, baboon-envelope pseudotyped lentiviral vectors (LV) outperformed VSVG-LVs for Nectin-4 CAR expressing in {beta} T cells resulting in an efficient anti-tumoral response in PDX mice.

Abstract/SummaryImmune checkpoint blockade can produce long-lasting responses in patients with metastatic melanoma; notably, combined CTLA-4/PD-1 blockade has been associated with approximately 52% melanoma specific 10-year survival (1). Yet, nearly half of patients experience minimal clinical benefit, and intensified regimens come with substantial risk of severe immune-related toxicity. The precise determinants of immunotherapy response are incompletely defined, reflecting a complex interplay between tumor biology and host immunity. This is especially consequential for patients whose disease progresses on checkpoint blockade, for whom effective salvage options are limited. In a series of patients with NRAS-mutated melanoma refractory to checkpoint inhibitors, we found that intratumoral administration of talimogene laherparepvec (T-VEC) combined with MEK inhibitor binimetinib induced exceptional clinical responses by amplification of pre-existing T cell responses and induction of de novo tumor-reactive immunity.

The major challenge in developing an effective adoptive cancer immunotherapy, is the ex-vivo generation of tumor-reactive cells, in sufficient numbers and with enhanced cytotoxic potential. It was recently demonstrated, that culturing of activated murine CD8+ T-cells on a "Synthetic Immune Niche" (SIN), consisting of immobilized CCL21 and ICAM-1, enhances T-cell expansion, increases cytotoxicity against cultured cancer cells and suppresses tumor growth in vivo [1, 2]. In the study reported here, we have tested the effect of the CCL21+ICAM1 SIN, on the expansion and cytotoxic phenotype of Tumor Infiltrating Lymphocytes (TIL), following activation with immobilized anti-CD3/CD28 stimulation, or commercial activation beads. The majority of TIL tested, displayed higher expansion when cultured on the coated SIN compared to cells incubated on uncoated substrate. Comparable enhancement of TIL proliferation was obtained by the CCL21+ICAM1 SIN, in a clinical setting that includes a 14-day rapid expansion procedure (REP), initiated with feeder cells, anti CD3 and IL-2. Co-incubation of post-REP TIL with matching target cancerous cells, demonstrated increased IFN{gamma} secretion beyond baseline in most of the TILs and a significant increase in granzyme B levels following activation on SIN. The SIN did not significantly alter the relative frequency of CD8/CD4 populations, as well as the expression of CD28, CD25 and several exhaustion markers. These results demonstrate the potential capacity of the CCL21+ICAM1 SIN to reinforce TIL-based immunotherapy.

Patient derived tumor organoids (PDTOs) have become relevant pre-clinical models for therapeutic modelling since they highly recapitulate patients response to treatment. Nevertheless, their value for immunotherapy modelling has not been fully explored. We developed a tumor processing protocol that enable the establishment of PDTOs and tumor infiltrating lymphocytes (TILs) isolation. By the optimization of functional assays, we compared the T-cells effector functions of matching PBMCs and TILs, demonstrating that PBMCs after co-culture and TILs after initial expansion display similar responses. In addition, the evaluation of cytokine production by fluorospot in combination with an image-based killing assay enable the screening of different immune-checkpoint inhibitors as well as its combination with target inhibitors. Our proof-of-concept functional assays showed the potential and versatility of PDTOs and T-cells co-culture systems for immunotherapy screening. The optimization of scalable functional assays downstream co-culture represents a significant step forward to increase the value of PDTOs as pre-clinical models for immunotherapeutic screens.