Supplementary Tables S1, S2 and S3

Bacillus Calmette-Guérin (BCG) instillations for the treatment of non-muscle-invasive bladder cancer patients can result in significant side effects and treatment failure. Immune checkpoint blockade and/or decreasing tumor-infiltrating myeloid suppressor cells may be alternative or complementary treatments. Here, we have characterized immune cell infiltration and chemoattractant molecules in mouse orthotopic MB49 bladder tumors. Our data show a 100-fold increase in CD45+ immune cells from day 5 to day 9 tumors including T cells and mainly myeloid cells. Both monocytic myeloid-derived suppressor-cells (M-MDSC) and polymorphonuclear (PMN)-MDSC were strongly increased in day 9 tumors, with PMN-MDSC representing ca. 70% of the myeloid cells in day 12 tumors, while tumor associated macrophages (TAM) were only modestly increased. The kinetic of PD-L1 tumor expression correlated with published data from patients with PD-L1 expressing bladder tumors and with efficacy of anti-PD-1 treatment, further validating the orthotopic MB49 bladder-tumor model as suitable for designing novel therapeutic strategies. Comparison of chemoattractants expression during MB49 bladder tumors grow highlighted CCL8 and CCL12 (CCR2-ligands), CCL9 and CCL6 (CCR-1-ligands), CXCL2 and CXCL5 (CXCR2-ligands), CXCL12 (CXCR4-ligand) and antagonist of C5/C5a as potential targets to decrease myeloid suppressive cells. Data obtained with a single CCR2 inhibitor however showed that the complex chemokine crosstalk would require targeting multiple chemokines for anti-tumor efficacy.

Protein sequences of all recombinant fusion proteins described in the manuscript

Time-lapse fluorescence microscopy of CMV-pp65-specific T cell mediated killing of MCSP+ tumor cells (Colo38) incubated with 15nM of EBV-pMHCI-IgG as control. Constructs are indicated in the respective panel. T cells are stained in red (PKH-26, Sigma), tumor cells in green (CMFDA, LifeTechnologies). Effector to target ratio was 3:1. Real-time is indicated in the lower left corner in h:m:s:ms (h=hours, m=minutes, s=seconds, ms=milliseconds), scale bar is shown in the lower right corner 0-50 µm (µm=micrometre).

Time-lapse fluorescence microscopy of CMV-pp65-specific T cell mediated killing of MCSP+ tumor cells (Colo38) incubated with 15nM of CMV-pMHCI-IgG. Constructs are indicated in the respective panel. T cells are stained in red (PKH-26, Sigma), tumor cells in green (CMFDA, LifeTechnologies). Effector to target ratio was 3:1. Real-time is indicated in the lower left corner in h:m:s:ms (h=hours, m=minutes, s=seconds, ms=milliseconds), scale bar is shown in the lower right corner 0-50 µm (µm=micrometre).

Supplementary Figure from Efficacy of CD40 Agonists Is Mediated by Distinct cDC Subsets and Subverted by Suppressive Macrophages

While regulatory T cells (Tregs) and macrophages have been recognized as key orchestrators of cancer-associated immunosuppression, their cellular crosstalk within tumors has been poorly characterized. Here, using spontaneous models for breast cancer, we demonstrate that tumor-associated macrophages (TAMs) contribute to the intratumoral accumulation of Tregs by promoting the conversion of conventional CD4+ T cells (Tconvs) into Tregs. Mechanistically, two processes were identified that independently contribute to this process. While TAM-derived TGF-β directly promotes the conversion of CD4+ Tconvs into Tregs in vitro, we additionally show that TAMs enhance PD-1 expression on CD4+ T cells. This indirectly contributes to the intratumoral accumulation of Tregs, as loss of PD-1 on CD4+ Tconvs abrogates intratumoral conversion of adoptively transferred CD4+ Tconvs into Tregs. Combined, this study provides insights into the complex immune cell crosstalk between CD4+ T cells and TAMs in the tumor microenvironment of breast cancer, and further highlights that therapeutic exploitation of macrophages may be an attractive immune intervention to limit the accumulation of Tregs in breast tumors.

ABSTRACTAgonistic αCD40 therapy has shown to inhibit cancer progression, but only in a fraction of patients. Hence, understanding the cancer cell-intrinsic and microenvironmental determinants of αCD40 therapy response is crucial to identify responsive patient populations and design efficient combination treatments. Here, we showed that the therapeutic efficacy of αCD40 in responder melanoma tumours, relied on pre-existing cDC1-primed CD8+ T cells, however cDC1s were dispensable after αCD40 administration. Surprisingly, in response to αCD40 the abundance of activated cDCs, potentially derived from cDC2s increased, thereby further activating antitumour CD8+ T cells. Hence, distinct cDC subsets are required to induce αCD40 responses. By contrast, lung tumours, characterised by a high abundance of macrophages, were resistant to αCD40 therapy. Combining αCD40 therapy with macrophage depletion led to tumour growth inhibition only in the presence of strong neoantigens. Accordingly, treatment with immunogenic cell-death inducing chemotherapy sensitised non-immunogenic tumours to αCD40 therapy.

The treatment of glioblastoma remains a challenge. Novel therapeutic strategies are urgently needed. Targeting the immunosuppressive glioblastoma-associated microenvironment is an interesting approach in this regard. Tumor-associated macrophages represent a distinct population of tumor-infiltrating immune cells with tumor-promoting features. The colony stimulating factor-1/ colony stimulating factor-1 receptor (CSF-1/CSF1R) axis plays an important role for macrophage differentiation and survival inside the tumor microenvironment. We thus aimed at investigating the antiglioma activity of CSF1R inhibition alone or in combination with blockade of programmed death (PD) 1. We detected CSF1R expression in paired tissue samples of primary and corresponding progressive glioblastoma. We investigated anti-CSF1R treatment alone or in combination with anti-PD1 antibodies in the orthotopic syngeneic VM/Dk SMA560 glioma mouse model, evaluated post-treatment effects and assessed treatment-induced cytotoxicity in a coculture model of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) ex vivo. Anti-CSF1R monotherapy increased the latency until the onset of neurological symptoms. Combinations of anti-CSF1R and anti-PD1 antibodies prolonged the latency until the onset of neurological symptoms and led to long-term survivors in vivo. Immunohistochemical analysis of post-treatment SMA-560 glioma revealed a modulation of the microenvironment, including increased T cell infiltration and reduced numbers of tumor-associated macrophages. Furthermore, we observed treatment-induced cytotoxicity of combined anti-CSF1R and anti-PD1 treatment in the PDM/TILs cocultures ex vivo. Taken together, our data indicates that CSF1R is a promising therapeutic target for glioblastoma, potentially in combination with PD1 inhibition.

Abstract Immune checkpoint blockade (ICB) with PD-1 or PD-L1 antibodies has been approved for the treatment of non-small cell lung cancer (NSCLC), often in combination with chemotherapy. However, only a minority of the patients respond and sustained remissions are rare. Anti-angiogenic therapy improves tumor response to ICB in mouse models of cancer and in patients with EGFR-mutant NSCLC, but the contribution of anti-angiogenic therapy to the clinical efficacy of ICB in patients with KRAS-mutant NSCLC remains unclear. We used genetically engineered mouse models of KrasG12D/p53null NSCLC, including a mismatch repair-deficient variant (KrasG12D/p53null/Msh2null) with higher mutational burden, and longitudinal imaging of individual tumors to study tumor response and resistance to combinations of ICB, anti-angiogenic therapy, and cisplatin chemotherapy. We found that anti-angiogenic blockade of VEGFA and angiopoietin-2 (ANGPT2) with the bispecific antibody A2V was superior to single VEGFA inhibition and markedly slowed tumor progression. But, contrary to findings in other tumor models, the addition of a PD1 antibody to A2V did not provide additive benefits and even accelerated growth of a fraction of the tumors. We implicated tumor-associated macrophages (TAMs) and PD-1+regulatory T cells (T-regs) in this growth-promoting response, and identified a pre-clinical strategy involving the differential targeting of two distinct TAM subsets that unleashed the therapeutic potential of anti-angiogenic immunotherapy and achieved regression of more than 80% of the lung tumors. Citation Format: Amaia Martinez-Usatorre, Ece Kadioglu, Chiara Cianciaruso, Alan Guichard, Bruno Torchia, Sina Nassiri, Martina Schmittnaegel, Carola H. Ries, Etienne Meylan, Ioanna Keklikoglou, Michele De Palma. Determinants of tumor resistance to anti-angiogenic immunotherapy in mouse models of Kras-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 75.

Glioblastoma is an aggressive primary tumor of the central nervous system. Targeting the immunosuppressive glioblastoma-associated microenvironment is an interesting therapeutic approach. Tumor-associated macrophages represent an abundant population of tumor-infiltrating host cells with tumor-promoting features. The colony stimulating factor-1/ colony stimulating factor-1 receptor (CSF-1/CSF1R) axis plays an important role for macrophage differentiation and survival. We thus aimed at investigating the antiglioma activity of CSF1R inhibition alone or in combination with blockade of programmed death (PD) 1. We investigated combination treatments of anti-CSF1R alone or in combination with anti-PD1 antibodies in an orthotopic syngeneic glioma mouse model, evaluated post-treatment effects and assessed treatment-induced cytotoxicity in a coculture model of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) ex vivo. Anti-CSF1R monotherapy increased the latency until the onset of neurological symptoms. Combinations of anti-CSF1R and anti-PD1 antibodies led to longterm survivors in vivo. Furthermore, we observed treatment-induced cytotoxicity of combined anti-CSF1R and anti-PD1 treatment in the PDM/TILs cocultures ex vivo. Our results identify CSF1R as a promising therapeutic target for glioblastoma, potentially in combination with PD1 inhibition.

Lung cancer resistance to PD-1 inhibition can be overcome by targeting tumor-associated macrophages in mouse models.

Colony-stimulating factor 1 receptor (CSF1R) blockade abates tumor-associated macrophage (TAM) infiltrates and provides marked clinical benefits in diffuse-type tenosynovial giant cell tumors. However, facial edema is a common adverse event associated with TAM elimination in patients. In this study, we examined molecular and cellular events associated with edema formation in mice and human patients with cancer treated with a CSF1R blocking antibody. Extended antibody treatment of mice caused marked body weight gain, an indicator of enhanced body fluid retention. This was associated with an increase of extracellular matrix-remodeling metalloproteinases (MMPs), namely MMP2 and MMP3, and enhanced deposition of hyaluronan (HA) and proteoglycans, leading to skin thickening. Discontinuation of anti-CSF1R treatment or blockade of MMP activity restored unaltered body weight and normal skin morphology in the mice. In patients, edema developed at doses well below the established optimal biological dose for emactuzumab, a CSF1R dimerization inhibitor. Patients who developed edema in response to emactuzumab had elevated HA in peripheral blood. Our findings indicate that an early increase of peripheral HA can serve as a pharmacodynamic marker for edema development and suggest potential interventions based on MMP inhibition for relieving periorbital edema in patients treated with CSF1R inhibitors.

Abstract Bladder cancer is one of the most common malignancies and has poor prognosis for patients with locally advanced, muscle-invasive, disease despite the efficacy of immune checkpoint blockade. To develop more effective immunotherapy strategies, we studied a genetic mouse model carrying deletion of Tp53 and Pten in the bladder, which recapitulates bladder cancer tumorigenesis and gene expression patterns found in patients. We discovered that tumor cells became more malignant and the tumor immune microenvironment evolved from an inflammatory to an immunosuppressive state. Accordingly, treatment with anti-PD1 was ineffective, but resistance to anti-PD1 therapy was overcome by combination with a CD40 agonist (anti-CD40), leading to strong antitumor immune responses. Mechanistically, this combination led to CD8+ T-cell recruitment from draining lymph nodes. CD8+ T cells induced an IFNγ-dependent repolarization toward M1-like/IFNβ-producing macrophages. CD8+ T cells, macrophages, IFN I, and IFN II were all necessary for tumor control, as demonstrated in vivo by the administration of blocking antibodies. Our results identify essential cross-talk between innate and adaptive immunity to control tumor development in a model representative of anti-PD1–resistant human bladder cancer and provide scientific rationale to target CD40 in combination with blocking antibodies, such as anti-PD1/PD-L1, for muscle-invasive bladder cancer.

Simulating a viral infection in tumor cells is an attractive concept to eliminate tumor cells. We previously reported the molecular design and the in vitro potency of recombinant monoclonal antibodies fused to a virus-derived peptide MHC class I complex that bypass the peptide processing and MHC loading pathway and directly displays a viral peptide in an MHC class I complex on the tumor cell surface. Here, we show that a vaccination-induced single peptide-specific CD8 T cell response was sufficient to eliminate B16 melanoma tumor cells in vivo in a fully immunocompetent, syngeneic mouse tumor model when mice were treated with mouse pMHCI-IgGs fusion proteins targeting the mouse fibroblast activation protein. Tumor growth of small, established B16 lung metastases could be controlled. The pMHCI-IgG had similar potency as an analogous pan-CD3 T-cell bispecific antibody. In contrast to growth control of small tumors, none of the compounds controlled larger solid tumors of MC38 cancer cells, despite penetration of pMHCI-IgGs into the tumor tissue and clear attraction and activation of antigen-specific CD8 T cells inside the tumor. pMHCI-IgG can have a similar potency as classical pan-T-cell recruiting molecules. The results also highlight the need to better understand immune suppression in advanced solid tumors.

Cancer immunotherapies are increasingly combined with targeted therapies to improve therapeutic outcomes. We show that combination of agonistic anti-CD40 with antiangiogenic antibodies targeting 2 proangiogenic factors, vascular endothelial growth factor A (VEGFA) and angiopoietin 2 (Ang2/ANGPT2), induces pleiotropic immune mechanisms that facilitate tumor rejection in several tumor models. On the one hand, VEGFA/Ang2 blockade induced regression of the tumor microvasculature while decreasing the proportion of nonperfused vessels and reducing leakiness of the remaining vessels. On the other hand, both anti-VEGFA/Ang2 and anti-CD40 independently promoted proinflammatory macrophage skewing and increased dendritic cell activation in the tumor microenvironment, which were further amplified upon combination of the 2 treatments. Finally, combined therapy provoked brisk infiltration and intratumoral redistribution of cytotoxic CD8(+) T cells in the tumors, which was mainly driven by Ang2 blockade. Overall, these nonredundant synergistic mechanisms endowed T cells with improved effector functions that were conducive to more efficient tumor control, underscoring the therapeutic potential of antiangiogenic immunotherapy in cancer.

Colony-stimulating factor 1 (CSF1) is a key regulator of monocyte/macrophage differentiation that sustains the protumorigenic functions of tumor-associated macrophages (TAMs). We show that CSF1 is expressed in human melanoma, and patients with metastatic melanoma have increased CSF1 in blood compared to healthy subjects. In tumors, CSF1 expression correlated with the abundance of CD8(+) T cells and CD163(+) TAMs. Human melanoma cell lines consistently produced CSF1 after exposure to melanoma-specific CD8(+) T cells or T cell-derived cytokines in vitro, reflecting a broadly conserved mechanism of CSF1 induction by activated CD8+ T cells. Mining of publicly available transcriptomic data sets suggested co-enrichment of CD8(+) T cells with CSF1 or various TAM-specific markers in human melanoma, which was associated with nonresponsiveness to programmed cell death protein 1 (PD1) checkpoint blockade in a smaller patient cohort. Combination of anti-PD1 and anti-CSF1 receptor (CSF1R) antibodies induced the regression of BRAF(V600E)-driven, transplant mouse melanomas, a result that was dependent on the effective elimination of TAMs. Collectively, these data implicate CSF1 induction as a CD8(+) T cell-dependent adaptive resistance mechanism and show that simultaneous CSF1R targeting may be beneficial in melanomas refractory to immune checkpoint blockade and, possibly, other T cell-based therapies.

Depletion of immunosuppressive tumor-associated macrophages (TAMs) or reprogramming toward a proinflammatory activation state represent different strategies to therapeutically target this abundant myeloid population. In this study, we report that inhibition of colony-stimulating factor-1 receptor (CSF-1R) signaling sensitizes TAMs to profound and rapid reprogramming in the presence of a CD40 agonist before their depletion. Despite the short-lived nature of macrophage hyperactivation, combined CSF-1R+CD40 stimulation of macrophages is sufficient to create a proinflammatory tumor milieu that reinvigorates an effective T cell response in transplanted tumors that are either responsive or insensitive to immune checkpoint blockade. The central role of macrophages in regulating preexisting immunity is substantiated by depletion experiments, transcriptome analysis of ex vivo sorted TAMs, and gene expression profiling of whole tumor lysates at an early treatment time point. This approach enabled the identification of specific combination-induced changes among the pleiotropic activation spectrum of the CD40 agonist. In patients, CD40 expression on human TAMs was detected in mesothelioma and colorectal adenocarcinoma.