anti-PD1 Therapy Research Articles

Mechanisms of Anti-PD Therapy Resistance in Digestive System Neoplasms.

Digestive system neoplasms are highly heterogeneous and exhibit complex resistance mechanisms that render anti-programmed cell death protein (PD) therapies poorly effective. The tumor microenvironment (TME) plays a pivotal role in tumor development, apart from supplying energy for tumor proliferation and impeding the body's anti-tumor immune response, the TME actively facilitates tumor progression and immune escape via diverse pathways, which include the modulation of heritable gene expression alterations and the intricate interplay with the gut microbiota. In this review, we aim to elucidate the mechanisms underlying drug resistance in digestive tumors, focusing on immune-mediated resistance, microbial crosstalk, metabolism, and epigenetics. We will highlight the unique characteristics of each digestive tumor and emphasize the significance of the tumor immune microenvironment (TIME). Furthermore, we will discuss the current therapeutic strategies that hold promise for combination with cancer immune normalization therapies. This review aims to provide a thorough understanding of the resistance mechanisms in digestive tumors and offer insights into potential therapeutic interventions.

Anatomical classification of advanced biliary tract cancer predicts programmed cell death protein 1 blockade efficacy.

Immune checkpoint blockade (ICB)-based immunotherapy has inspired new hope for advanced biliary tract cancer (BTC) treatment; however, there are no prior studies that primarily focus on different anatomical types of unresectable BTCs reacting differently to ICB. We retrospectively collected data on advanced BTC patients who received anti-programmed cell death protein 1 (anti-PD1) therapy from two affiliated hospitals of Sun Yat-Sen university. The effects of anti-PD1 were compared for different anatomical sites. The GSE32225 and GSE132305 datasets were used to further analyze differences in the immune microenvironments between intrahepatic cholangiocarcinoma (ICC) and extrahepatic cholangiocarcinoma (ECC). A total of 198 advanced BTC patients were enrolled in this study, comprising 142 patients with ICC and 56 with other cancer types ("Others" group), including ECC and gallbladder cancer. In the anti-PD1 treated patients, the ICC group (n = 90) achieved longer median progression-free survival (mPFS) (9.5 vs. 6.2 months, p = 0.02) and median overall survival (mOS) (15.1 vs. 10.7 months, p = 0.02) than the Others group (n = 26). However, chemotherapy did not show different effects between the two groups (mOS: 10.6 vs. 12.1 months, p = 0.20; mPFS: 4.9 vs. 5.7months, p = 0.83). For the first-line anti-PD1 therapy, the ICC group (n = 70) achieved higher mOS (16.0 vs. 11.8months, p = 0.04) than the Others group (n = 19). Moreover, most chemokines, chemokine receptors, major histocompatibility complex molecules, immunostimulators, and immunoinhibitors were stronger in ICC than ECC; furthermore, CD8+ T cells and M1 macrophages were higher in ICC than ECC for most algorithms. The immune differential genes were mainly enriched in antigen processing and presentation as well as the cytokine receptors. This study shows that the efficacy of anti-PD1 therapy was higher in ICC than in other types of BTCs. Differences in the immune-related molecules and cells between ICC and ECC indicate that ICC could benefit more from immunotherapy.

CRP and soluble CTLA4 are determinants of anti-PD1 resistance in gastrointestinal cancer.

Targeting immune inhibitory checkpoint (IC) pathways have attracted great attention as a promising strategy for treating gastrointestinal (GI) cancer. However, the therapeutic efficacy is low in most cases, and little progress has been made in establishing biomarkers that predict the possible responses, and combination regimens that enhance the therapeutic efficacy. As a predictive biomarker, soluble forms of IC molecules have been recently highlighted. However, little is known about which IC molecules is most critically associated with the treatment resistance, and also about the biological and immunological roles of the IC molecules in GI cancer. In this study, we analyzed sera obtained from advanced gastric cancer patients before and one month after treatment with anti-PD1 nivolumab for soluble IC molecules by ELISA. We found that decrease of soluble CTLA4 (sCTLA4) at posttreatment were significantly associated with a better prognosis, and combination with low level of CRP at posttreatment more clearly defined anti-PD1 responders with long-term survival. Indeed, in the in vitro setting, CRP stimulation upregulated CTLA4 expression in tumor cells followed by generation of sCTLA4 that suppressed CTL induction, and simultaneously conferred high self-renewal and invasive abilities on the tumor cells accompanied by increase of EMT-related gene expressions. In the in vivo setting, CRP injection elevated sCTLA4 level in sera of mouse tumor metastasis models, leading to failure of anti-PD1 therapy. However, treatment with anti-CTLA4 mAb or a PPARγ agonist that can reduce in vivo CRP successfully elicited anti-tumor efficacy in the anti-PD1 resistant models. These suggest that targeting CRP and sCTLA4 may be a promising strategy for improving clinical outcomes in the treatments, including anti-PD1 therapy, of GI cancer.

Interferon-stimulated neutrophils as a predictor of immunotherapy response

Despite the remarkable success of anti-cancer immunotherapy, its effectiveness remains confined to a subset of patients-emphasizing the importance of predictive biomarkers in clinical decision-making and further mechanistic understanding of treatment response. Current biomarkers, however, lack the power required to accurately stratify patients. Here, we identify interferon-stimulated, Ly6Ehi neutrophils as a blood-borne biomarker of anti-PD1 response in mice at baseline. Ly6Ehi neutrophils are induced by tumor-intrinsic activation of the STING (stimulator of interferon genes) signaling pathway and possess the ability to directly sensitize otherwise non-responsive tumors to anti-PD1 therapy, in part through IL12b-dependent activation of cytotoxic Tcells. By translating our pre-clinical findings to a cohort of patients with non-small cell lung cancer and melanoma (n= 109), and to public data (n= 1440), we demonstrate the ability of Ly6Ehi neutrophils to predict immunotherapy response in humans with high accuracy (average AUC≈ 0.9). Overall, our study identifies a functionally active biomarker for use in both mice and humans.

Efficiency and safety of hepatic arterial infusion chemotherapy (HAIC) combined with anti-PD1 therapy versus HAIC monotherapy for advanced hepatocellular carcinoma: A multicenter propensity score matching analysis.

To investigate the clinical efficacy and safety of combination therapy of hepatic arterial infusion chemotherapy (HAIC) and anti-programmed cell death protein-1 (PD-1) therapy in the treatment of advanced hepatocellular carcinoma (HCC). In this retrospective clinical research, from March 2018 to December 2019, 1158 HCC patients categorized as BCLC stage C were reviewed for eligibility. We utilized propensity score matching (PSM) to mitigate initial disparities between the groups. The evaluation of the best tumor response was conducted in accordance with mRECIST 1.1 criteria. The difference in survival outcomes including overall survival (OS), progression-free survival (PFS), and objective response rate (ORR) between groups were compared. Following the eligibility review, 453 patients underwent a combined treatment of HAIC with PD1 inhibitors (HAIC-PD1 group), while 221 patients received HAIC monotherapy (HAIC group) met the inclusion criteria and were finally enrolled in this study. In the entire cohort, the HAIC-PD1 group exhibited significantly prolonged overall survival (median overall survival: 40.4 months vs. 9.7 months, p < 0.001) and progression-free survival (median progression-free survival: 22.1 months vs. 5.8 months, p < 0.001). By propensity score, patients were matched according to baseline differences, resulting in all 442 patients in group HAIC-PD1 (n = 221) and group HAIC (n = 221). After PSM adjustment, as well, the survival of the HAIC-PD1 group was still distinctly longer than the HAIC group (median overall survival time, 40.4 months vs 9.7 months, p < 0.001; median progression-free survival, 22.1 months vs 5.7 months, p < 0.001). Univariate and multivariable analysis demonstrated that AFP level, metastasis, and therapeutic schedule were independent predictive factors for overall survival. The combination therapy of HAIC and PD1 inhibitors successfully extended OS to advanced HCC patients and could be a better choice than HAIC monotherapy.

Ct-based subregional radiomics using hand-crafted and deep learning features for prediction of therapeutic response to anti-PD1 therapy in NSCLC

PurposeTo develop and externally validate subregional radiomics for predicting therapeutic response to anti-PD1 therapy in non-small-cell lung cancer (NSCLC). MethodsSixty-six patients from center 1 served as training and internal validation cohorts. Thirty patients from center 2 and thirty patients from center 3 served as external validation 1 and external validation 2 cohorts, respectively. The lesions identified on CT scans were subdivided into two phenotypically consistent subregions by automatic clustering on the patient-level and population-level (denoted as marginal S1 and inner S2). Handcrafted and deep learning-based features were extracted separately from the entire tumor region and subregions, then selected using the intraclass correlation coefficient and least absolute shrinkage and selection operator regression (LASSO). Radiomics signatures (RSs) were built integrating the selected features and correlation coefficients using a logistic regression method. Area under the receiver operating characteristic (ROC) curve (AUC) was calculated to assess the RSs. ResultsRSs derived from S1 outperformed those from S2 and the whole tumor region for both handcrafted and deep learning features. The Fusion-RS incorporating the two feature types achieved the best prediction performance in training (AUC = 0.947, 95 % Confidence Interval [CI] 0.905–0.989, SPE = 0.895, SEN = 0.878), internal validation (AUC = 0.875, 95 % CI: 0.782–0.969, SPE = 0.724, SEN = 0.952), external validation 1 (AUC = 0.836, 95 % CI: 0.694–0.977, SPE = 1.000, SEN = 0.533) and external validation 2 (AUC = 0.783, 95 % CI: 0.613–0.953, SPE = 0.765, SEN = 0.692) cohorts. ConclusionsSubregional radiomics analysis can be useful for predicting therapeutic response to anti-PD1 therapy. The developed Fusion-RS may be considered as a potential non-invasive tool for individual treatment managements.

Clinical and therapeutical significances of the cluster and signature based on oxidative stress for osteosarcoma.

It is of great clinical significance to find out the ideal tumor biomarkers and therapeutic targets to improve the prognosis of patients with osteosarcoma (OS). Oxidative stress (OXS) can directly target intracellular macromolecules and exhibit dual effects of tumor promotion and suppression. OXS-related genes (OXRGs) were extracted from public databases, including TARGET and GEO. Univariate Cox regression analysis, Random Survival Forest algorithm, and LASSO regression were performed to identify prognostic genes and establish the OXS-signature. The efficacy of the OXS-signature was further evaluated by Kaplan-Meier curves and timeROC package. Evaluation of immunological characteristics was achieved based on ESTIMATE algorithm and ssGSEA. Submap algorithm was used to explore the response to anti-PD1 and anti-CTLA4 therapy for OS. Drug response prediction was conducted by using pRRophetic package. The expression values of related genes in the OXS-signature were detected with PCR assays. Two OXS-clusters were identified for OS, with remarkable differences of clusters presented in prognosis. Kyoto Encyclopedia of Genes Genomes (KEGG) analysis showed that differentially expressed genes (DEGs) between the OXS-clusters were significantly enriched in several immune-related pathways. Patients with lower OS-scores attained better clinical outcomes, and presented more sensitivity to ICB therapy. By contrast, OS patients with higher OS-scores revealed more sensitivity to certain drugs. Furthermore, critical genes, RHBDL2 and CGREF1 from the model, were significantly higher expressed in OS cell lines. Our study identified the clusters and signature based on OXS, which would lay the foundation for molecular experimental research, disease prevention and treatment of OS.

Kinase Insert Domain Receptor Q472H Pathogenic Germline Variant Impacts Melanoma Tumor Growth and Patient Treatment Outcomes.

We previously reported a higher incidence of a pathogenic germline variant in the kinase insert domain receptor (KDR) in melanoma patients compared to the general population. Here, we dissect the impact of this genotype on melanoma tumor growth kinetics, tumor phenotype, and response to treatment with immune checkpoint inhibitors (ICIs) or targeted therapy. The KDR genotype was determined and the associations between the KDR Q472H variant (KDR-Var), angiogenesis, tumor immunophenotype, and response to MAPK inhibition or ICI treatment were examined. Melanoma B16 cell lines were transfected with KDR-Var or KDR wild type (KDR-WT), and the differences in tumor kinetics were evaluated. We also examined the impact of KDR-Var on the response of melanoma cells to a combination of VEGFR inhibition with MAPKi. We identified the KDR-Var genotype in 81/489 (37%) patients, and it was associated with a more angiogenic (p = 0.003) and immune-suppressive tumor phenotype. KDR-Var was also associated with decreased PFS to MAPKi (p = 0.022) and a trend with worse PFS to anti-PD1 therapy (p = 0.06). KDR-Var B16 murine models had increased average tumor volume (p = 0.0027) and decreased CD45 tumor-infiltrating lymphocytes (p = 0.0282). The anti-VEGFR treatment Lenvatinib reduced the tumor size of KDR-Var murine tumors (p = 0.0159), and KDR-Var cells showed synergistic cytotoxicity to the combination of dabrafenib and lenvatinib. Our data demonstrate a role of germline KDR-Var in modulating melanoma behavior, including response to treatment. Our data also suggest that anti-angiogenic therapy might be beneficial in patients harboring this genotype, which needs to be tested in clinical trials.

TTK inhibition activates STING signal and promotes anti-PD1 immunotherapy in breast cancer

Despite progress in the application of checkpoint immunotherapy against various tumors, attempts to utilize immune checkpoint blockade (ICB) agents in triple negative breast cancer (TNBC) have yielded limited clinical benefits. The low overall response rate of checkpoint immunotherapy in TNBC may be attributed to the immunosuppressive tumor microenvironment (TME). In this study, we investigated the role of mitogen-associated kinase TTK in reprogramming immune microenvironment in TNBC. Notably, TTK inhibition by BAY-1217389 induced DNA damage and the formation of micronuclei containing dsDNA in the cytosol, resulting in elicition of STING signal pathway and promoted antitumor immunity via the infiltration and activation of CD8+ T cells. Moreover, TTK inhibition also upregulated the expression of PD-L1, demonstrating a synergistic effect with anti-PD1 therapy in 4T1 tumor-bearing mice. Taken together, TTK inhibition facilitated anti-tumor immunity mediated by T cells and enhanced sensitivity to PD-1 blockade, providing a rationale for the combining TTK inhibitors with immune checkpoint blockade in clinical trials.

Pyroptosis-Related Subtypes Predict the Response of Clear Cell Renal Cell Carcinoma to Targeted Therapy.

Pyroptosis plays a crucial role in anti-tumor immunity and in formation of the immune microenvironment. However, whether pyroptosis is involved in the progression of clear cell renal cell carcinoma (ccRCC) is still unclear. Personalized treatment of ccRCC requires detailed molecular classification to inform a specific therapy. Molecular subtyping of ccRCC was performed based on consensus clustering of pyroptosis-related genes. The characteristics of these molecular subtypes were explored at the genome, transcriptome and protein levels. Single-cell RNA sequencing and CIBERSORT analysis were used to analyse the immune microenvironment of ccRCC, while Lasso regression was used to develop a prediction model based on hub genes. Expression of the pyroptosis-related gene GSDMB was also investigated at the tissue and cellular levels. Two molecular subtypes were identified based on the clustering of pyroptosis-related genes. Cluster 1 was associated with activation of classical oncogenic pathways, especially the angiogenesis pathway. Cluster 2 was associated with activation of immune-related pathways and high levels of immunosuppressive cells, exhausted CD8+ T cells, and tumor-associated fibroblast infiltration. Clusters 1 and 2 were thus defined as the angiogenic and inflamed subtypes, respectively. The two subtypes were predictive of the response of ccRCC to anti-angiogenic therapy and immunotherapy, with Cluster 1 patients benefiting from anti-angiogenic therapy and Cluster 2 patients showing better response to anti-PD1 inhibitor therapy. Furthermore, a 9-gene expression signature (HJURP, NUF2, KIF15, MELK, TPX2, PLK1, CDCA3, CTLA4, FOXP3) was identified that could predict outcome and response to immune checkpoint blockade therapy in test cohorts. Finally, GSDMB was found to be involved in the development of renal clear cell carcinoma. These results on pyroptosis-related genes in ccRCC provide a theoretical basis for understanding molecular heterogeneity and for the development of individualized treatment strategies.

Abstract C074: VPS34 inhibition delays activation-induced STING degradation to prolong STING signaling and improve anti-tumor efficacy in preclinical models

Abstract Background: The STING/interferon (IFN) response pathway has been a target of cancer immunotherapy for many years, as a method for reactivating the anti-tumor immune response. Stimulation of the STING pathway leads to activation and infiltration of immune cells into the tumor, and cancer cell death. However, multiple STING agonists have failed in the clinic due to poor efficacy or toxicity1. STING activation leads to its degradation within a few hours through trafficking to the lysosome, as a mechanism to control immune cell hyperactivation2. Although this safety mechanism is important for preventing autoimmunity and chronic inflammation, it diminishes the continuous activation of immune-mediated cancer cell killing, especially in an immunosuppressive tumor microenvironment. VPS34 is a lipid kinase which plays an integral role in endosomal trafficking and autophagy3. Previous publications have demonstrated a role for VPS34 inhibition in increased effector immune cell infiltration, which synergizes with anti-PD1 therapy, demonstrating a link to immune activation4. Herein, we demonstrate that the in vivo combination efficacy with a VPS34 inhibitor and a STING agonist is due to reduced degradation of activated STING, through the inhibition of VPS34-mediated endosomal trafficking. Materials and Methods: Cancer cell lines were cultured using recommended medium. STING activity was measured by Western blot and via a CisBio pTBK1 kit. ADU-S100/MSA-2 were sourced from MedChemExpress. CCL5, CXCL10 and IFNb ELISA’s were purchased from R&amp;D systems. Primers used to measure mRNA expression were obtained from IDT. Results: DP-9244 is a potent and selective VPS34 inhibitor. The combination of DP-9244 with the STING agonist, ADU-S100, enhances IFN-mediated gene expression and cytokine secretion in the murine melanoma cell line, B16F10, and in the human and murine myeloid cell lines THP-1 and DC2.4, demonstrating combination activity in both cancer and immune cells. This combination activity is STING-dependent as demonstrated by the complete loss of IRF and NFκB activity in the THP-1 STING-/- cell line when treated with ADU-S100, DP-9244 or the combination. Mechanistically, VPS34 inhibition delays degradation of activated STING and prolongs downstream signaling of pTBK1 in multiple human and murine cancer cell lines. Importantly, the in vitro effects extend to in vivo efficacy in the B16F10 model, in which the combination of DP-9244 and ADU-S100 results in increased tumor growth control and number of mice responding to treatment. Conclusions: These data provide a strong preclinical rationale to combine a VPS34 inhibitor with a STING agonist as a method of improving STING pathway mediated anti-tumor efficacy.

Abstract A169: Overcoming tumor-associated immune suppression and resistance to cancer immunotherapy using Hyal2-ADCC

Abstract Background: Multiple clinical studies have addressed the role of immunotherapies for solid cancers. While these studies have demonstrated promising results and improvements over existing therapies, less than half of patients with advanced disease show the clinical benefits of immunotherapy. Furthermore, once tumors start to produce metastases, it becomes one of the leading causes of cancer-related mortality. Hyal2-ADC is a novel conjugate of anti-Hyal2 monoclonal antibody and cytotoxic payload PNU. Hyaluronidase 2 (Hyal2), which is a membrane-bound enzyme expressed by both MDSCs and epithelial tumor cells, serves as a molecular target for the Hyal2-ADC. Importantly, our anti-Hyal2 antibodies recognize both human and mouse Hyal2. Methods: Expression of Hyal2 in human and mouse cancer tissues was assessed using immunohistochemistry and Western Blotting. The in vitro effects of Hyal2-ADC tumor cells and MDSCs were evaluated using MTT assay and immunofluorescence. A single-cell RNA sequencing was utilized for the transcriptomic analysis of Hyal2-expressing myeloid cells. The in vivo anti-tumor activity of Hyal2-ADC was evaluated using syngeneic mouse tumor models and human xenograft tumor models in immunodeficient mice. Mice with established bladder or prostate tumors were used in this study. Results: Administration of Hyal-ADC alone led to the inhibition of tumor growth in a dose-dependent manner. Importantly, the therapeutic effect of Hyal2-ADC was associated with remodeling the tumor microenvironment, reduction of immunosuppressive myeloid cells, and influx of immune T cells. A combination of Hyal2-ADC with anti-PD1 therapy resulted in complete tumor eradication in mice with syngeneic established tumors (initial volume &amp;lt;200 mm3) in 100% treated mice. The cured mice demonstrated long-term protection from tumor re-challenge. Furthermore, the combination therapy of mice with large, advanced established tumors (&amp;gt;200mm3) resulted in 80% tumor eradication. Conclusions: Hyal2-ADC is a first-in-class drug that selectively targets Hyal2-expressing cells. The modulation of the immunosuppressive tumor microenvironment and development of efficient immune memory observed in murine models suggests that Hyal2-ADC alone or in combination with anti-PD1 therapy could be utilized for the treatment of solid tumors associated with deregulated HA metabolism, including bladder, prostate, breast, brain, and other cancers. Mechanistically, the enzyme Hyal2 is involved in the degradation of tumor-associated hyaluronan (HA), one of the major components of the extracellular matrix in tumor stroma. Depletion of Hyal2-expressing cells leads to normalization of HA metabolism in the tumor microenvironment supporting generation of T cell-mediated anti-tumor immune response. Overall, this study introduces a novel concept to remodeling the tumor microenvironment, bypassing tumor-associated immune suppression, and overcoming resistance to cancer immunotherapy, thus representing a novel therapeutic strategy for patients. Citation Format: Sergei Kusmartsev, William Donelan, Wayne Brisbane, Brianna Nguyen, Meng-Lun Hsieh, Shiwu Li, Paul Dominguez-Gutierrez, Padraic O'Malley, Paul L Crispen. Overcoming tumor-associated immune suppression and resistance to cancer immunotherapy using Hyal2-ADCC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A169.

Abstract C077: STC-15, a small molecule inhibitor of the RNA methyltransferase METTL3, activates anti-tumor immunity and reshapes the tumor microenvironment

Abstract Background METTL3 is the RNA methyltransferase responsible for the deposition of N-6-methyladenosine modification (m6A) on mRNA, to regulate its stability, splicing and protein translation. Small molecule inhibitors of METTL3 catalytic activity have previously demonstrated anti-tumor efficacy in pre-clinical models of acute myeloid leukemia (AML) and solid tumors. Inhibition of METTL3 was shown to induce a cell-intrinsic innate immune response through activation of the double strand RNA (dsRNA) sensing machinery and interferon (IFN) signalling. Here we present new pre-clinical data, investigating the combination of METTL3 inhibitor (METTL3i) with DNA damaging therapies. We show that METTL3 inhibition profoundly changes the tumor microenvironment (TME), and we further demonstrate the generation of durable immune memory in mice treated with METTL3i, with or without anti-PD1 therapy. Materials &amp; Methods The orally bioavailable small molecule clinical-stage METTL3i STC-15, or the equipotent tool compound STM3675 were used in these studies. To characterise transcriptomic changes following METTL3 inhibition, RNA sequencing was performed. Induction of specific genes was validated by Western Blot, and cytokine secretion assessed by ELISA. In-vitro cell viability was assessed by Cell TiterGlo (Promega). In vivo efficacy studies used subcutaneous MC38 and GL261 syngeneic tumor models, and characterisation of the TME was performed by flow cytometry. Results Treatment with METTL3i sensitized cells to DNA damaging agents such as doxorubicin. On the molecular level, further enhancement of the IFN pathway by the combination treatment was observed. Moreover, combination of METTL3i with radiation treatment (RT) in vivo in the MC38 colorectal syngeneic mouse model demonstrated a clear benefit of the combination over each of the single treatments. METTL3i alone or in combination with RT led to a major TME remodulation, reducing the number of Tregs, M2 macrophages and neutrophils, and increasing the number of M1 macrophages and CD8+ T-cells in tumors. The GL261 syngeneic glioma model is exquisitely sensitive to anti-PD1 treatment. Similarly, METTL3i treatment led to multiple complete regressions, and the combination of METTL3i and anti-PD1 further accelerated the occurrence of regressions. Following a 3-weeks’ observation period, tumor free mice were re-inoculated with GL261 cells. All tumor-free mice remained tumor free after the rechallenge. Conclusions In pre-clinical cancer models, treatment with METTL3i such as STC-15 resulted in activation of innate immune pathways and subsequent enhancement of cytotoxic T-cell activity. In-vivo, STC-15 had a profound effect on the TME, shifting it from an immunosuppressive, pro-tumorigenic state to an immunostimulatory, anti-tumor state. In combination with anti-PD1, METTL3 inhibitors generate a durable anti-tumor response and lasting immune memory. A First-in-Human dose escalating study evaluating STC-15 monotherapy in patients with solid tumors is ongoing (NCT05584111). Citation Format: Yaara Ofir-Rosenfeld, Lina Vasiliauskaitė, Joanna Obacz, Georgia Tsagkogeorga, Josefin-Beate Holz, Jerry McMahon, Oliver Rausch. STC-15, a small molecule inhibitor of the RNA methyltransferase METTL3, activates anti-tumor immunity and reshapes the tumor microenvironment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C077.

Abstract C167: An SN38 dendrimer nanoparticle, DEP irinotecan (SN38-SPL9111), demonstrates efficacy in mouse models of gastrointestinal cancer and augments anti-tumor effects of immune checkpoint blockade and PARP inhibition

Abstract Background: Irinotecan is a topoisomerase 1 inhibitor pro-drug used to treat gastrointestinal (GI) cancers including as first line for colorectal cancer (CRC) as part of the FOLFIRI plus monoclonal antibody (mAb) regimen, and for pancreatic cancer in the FOLFIRINOX regimen. However, irinotecan causes severe and dose-limiting side effects, including diarrhea and neutropenia. Starpharma has developed a highly optimized polylysine dendrimer enhanced (DEP) nanoparticle conjugate of the irinotecan active metabolite, SN38 (DEP SN38 or DEP irinotecan). DEP SN38 avoids the need for liver conversion of irinotecan to SN38 and achieves preferential tumor targeting. DEP SN38 is in phase 2 clinical investigation as monotherapy or combination therapy in patients with solid tumors (EudraCT no: 2019-001318-40). SN38 is known to enhance the efficacy of immune checkpoint blockade (ICB) via effects on immune cells in the tumor microenvironment. Here, we evaluated the anti-tumor effects of DEP SN38 alone or in combination with either ICB (anti- programmed cell death-1 [PD1] mAb) or poly adenosine diphosphate (ADP) ribose polymerase (PARP) inhibition (olaparib) in mouse models of GI cancer. Methods: HT29 (human CRC) and CAPAN-1 (human pancreatic cancer) cell line xenografts were established subcutaneously in BALB/c nude or NOD-scid-Gamma immunodeficient mice, respectively. Three doses of DEP SN38 (at 5-10mg/kg SN38) or irinotecan (35-80 mg/kg) were administered intravenously (IV) on days 1, 8, 15. Olaparib (50 mg/kg) was delivered by daily IV injection for 5 days/week over 3 weeks. DEP SN38 (IV on days 1, 8, 15 at 18-35 mg/kg SN38) or irinotecan (75 mg/kg) plus rat anti-PD1 mAb therapy or control mAb (intraperitoneally, 100 mg/kg then 50 mg/kg on days 5, 8, 12) was evaluated in both MC38 (C57BL/6) and CT26 (BALB/c) allograft models of CRC. Toleration of drug was bodyweight loss ≤15%. Results: DEP SN38 treatment of HT29 or CAPAN-1 xenografts led to tumor regressions, prolonged control of tumor growth and improved survival. In contrast, conventional irinotecan had minimal effect on tumor growth versus vehicle control. DEP SN38 was well tolerated by mice with minimal bodyweight loss. Administration of anti-PD1 mAb alone confirmed the known modest sensitivity of MC38, and resistance of CT26 syngeneic tumors, to ICB. DEP SN38 plus anti-PD1 mAb, enhanced anti-tumor responses in the MC38 model beyond those seen for each agent alone and also resulted in tumor eradication and prolonged survival. In the CT26 model, anti-PD1 mAb was also augmented by addition of DEP SN38 but not irinotecan alone, despite resistance of CT26 tumors to anti-PD1 mAb alone. The HT29 CRC model was resistant to Olaparib alone. In contrast, anti-tumor effects and prolonged survival were observed with olaparib plus DEP SN38. Conclusions: DEP SN38 demonstrated superior efficacy compared to irinotecan when used in models of GI cancer. The anti-tumor efficacy of both ICB and PARP inhibition was augmented by DEP SN38 in models of CRC, and these combinations should be investigated clinically. Citation Format: Benjamin J Blyth, Brian D Kelly, Michael Giannis, Anne Cargill, Aynaz Seta, Graham P Heery, Anthony Eglezos, Cameron N Johnstone, Jeremy R A Paull. An SN38 dendrimer nanoparticle, DEP irinotecan (SN38-SPL9111), demonstrates efficacy in mouse models of gastrointestinal cancer and augments anti-tumor effects of immune checkpoint blockade and PARP inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C167.

FLT3L-dependent dendritic cells control tumor immunity by modulating Treg and NK cell homeostasis

FLT3-L-dependent classical dendritic cells (cDCs) recruit anti-tumor and tumor-protecting lymphocytes. We evaluate cancer growth in mice with low, normal, or high levels of cDCs. Paradoxically, both low or high numbers of cDCs improve survival in mice with melanoma. In low cDC context, tumors are restrained by the adaptive immune system through influx of effector Tcells and depletion of Tregs and NK cells. High cDC numbers favor the innate anti-tumor response, with massive recruitment of activated NK cells, despite high Treg infiltration. Anti CTLA-4 but not anti PD-1 therapy synergizes with FLT3-L therapy in the cDCHi but not in the cDCLo context. A combination of cDC boost and Treg depletion dramatically improves survival of tumor-bearing mice. Transcriptomic data confirm the paradoxical effect of cDC levels on survival in several human tumor types. cDCHi-TregLo state in such patients predicts best survival. Modulating cDC numbers via FLT3 signaling may have therapeutic potential in human cancer.

Abstract IA001: Patient-derived tumor organoids to probe the cancer immune interface

Abstract The development of rational (combination) immunotherapies is limited by an incomplete understanding of what determines a tumor’s sensitivity to immune attack. Despite great heterogeneity in resistance mechanisms between patients, functional model systems to study cancer/immune cell interactions at the level of individual patients have been lacking. Here we highlight the potential of organoid/immune cell co-cultures as individualized models of anti-tumor immunity. First, we established a co-culture platform of autologous tumor organoids and peripheral blood lymphocytes from patients with mismatch-repair deficient colorectal cancer (dMMR-CRC) and non-small-cell lung cancer (NSCLC). This allows enriching tumor-reactive T cells from peripheral blood of individual patients, and creating personalized test systems to evaluate their capacity to kill matched tumor organoids. Second, subclonal immune escape is a major barrier to achieving complete responses to cancer immunotherapies. While most tumours consist of multiple genetically distinct clones, directly investigating the potential for immune escape at the clonal level has been impossible so far, due to challenges in isolating and propagating individual tumour subclones from human cancers for functional studies. By leveraging the multi-region TRACERx study, we established multi-region organoid libraries of &amp;gt;20 parallel sublines from patients with NSCLC. Co-cultures of each subline with autologous tumour infiltrating lymphocytes (TIL) reveal substantial heterogeneity in the capacity of individual clonal sublines to elicit a T-cell response. We show that subclonal immune escape can be mediated by antigen-dependent and -independent mechanisms. These results show (i) that tumour evolution can give rise to distinct cancer clones with intrinsic differences in immune evasion capacity and (ii) provide an approach to prospectively identify and isolate immune evading subclones from cancer patients. Third, analysis of a clinical cohort of patients treated with anti-PD1 therapy revealed that gamma delta T cells are critical mediators of the response in dMMR-CRC patients with genetic inactivation of beta-2-microglobulin (B2M). Co-cultures of organoids and gamma delta T cells provide direct evidence that loss of B2M increases reactivity of patient-derived gamma delta T cells towards tumor organoids. This highlights the need for functional models that move beyond conventional T cells. Future developments are directed towards establishment of complex co-culture systems to study how communication between organoids and immune cells is mediated by cell surface factors and secreted molecules, and how cellular crosstalk is altered in cancer. Citation Format: Krijn K Dijkstra. Patient-derived tumor organoids to probe the cancer immune interface [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr IA001.

Metabolic Reprograming of Exhausted Intratumoral CD8+ T-Cell Underlies Anti-Tumor Activity of Sumoylation Inhibitors in Large B Cell Lymphoma

Immune checkpoint inhibitors (ICI) targeting PD1 have revolutionized our approach to solid tumors and Hodgkin lymphoma, but they have not been effective in treating Large B Cell Lymphoma (LBCL). In prior work, we used single cell spatial profiling of human tumors to show that exhausted CD8 tumor and TREG populations were associated with PD1 resistance in LBCL (Colombo et al. Blood Adv 2022). Here we show, using a syngeneic model of LBCL, that PD1 resistance is associated with lower CD8+ T-cell basal metabolic rate (BMR) in tumor infiltrating lymphocytes (TIL), and that treatment with the covalent SUMOylation inhibitor (SB-4826) increases the metabolic rate of TIL by metabolic reprograming. Single cell metabolic profiling of human LBCL is on-going and will be presented as well. In summary these studies demonstrate that metabolic dysfunction of T cells in LBCL underlie resistance to PD1 checkpoint inhibitors and that this can be overcome by treatment with SUMOlyation inhibitors. Direct measurement of intra-tumoral metabolic state is difficult to perform on clinical samples, however large tumor size is hypothesized to be a surrogate for increased metabolic derangement. As tumors expand and become more metabolically active themselves, infiltrating immune cells experience decreased O2 tension as well as nutrient deprivation, ultimately leading to decreased anti-tumor immune responses. In support of this idea, it has been observed that high metabolic tumor volume at baseline predicts survival in LBCL patients, independent of their initial response to therapy. We have shown that mice with established (&amp;gt;125mm 3) A20 tumors are resistant to anti-PD1 therapy. However, A20 bearing mice treated ~5 days post implantation are responsive to anti-PD1 therapy. To understand these findings, we examined sensitive (small) and resistant (large) A20 tumors by flow cytometry analysis and found that resistant tumors had lower relative numbers of T cells, lower absolute numbers of CD8+ T cells, increased absolute numbers of TREG and a strikingly decreased CD8/TREG ratio. Additionally, the resistant tumors had increased absolute numbers of pre-exhausted and exhausted CD8+ T cells as well as decreased BMR. Small ubiquitin-like modifiers (SUMO) proteins regulate a variety of cellular processes in tumor and immune cells and inhibitors of SUMOylation have shown anti-tumor activity in a variety of preclinical tumor models with potential synergy with checkpoint inhibitors targeting PD-1 (Cannon et al. Cancer Res, 2023). We demonstrate that a dose of 100mg/kg of SB-4826 led to a complete response in all mice treated (Fig. 1A) and we also show that this effect is dependent on CD8+ T-cells and elicits a long term memory response, with 0 of 15 mice in remission developing tumor upon rechallenge. A low dose (25mg/kg) of SB-4826 failed to elicit a response in any of the mice treated while anti-PD1 therapy elicited a response in 1/6 mice (Fig. 1A). Combination of 25mg/kg SB-4826 with anti-PD1 therapy demonstrated synergism with a response in 3/6 mice (Fig. 1A). We also demonstrate that global SUMOylation levels are higher in PD1+ TIM3+ CD8+ TILs compared to PD1- TIM3- CD8+ T-cells, suggesting that SUMOylation may be acting as a negative regulator of T-cell function. Using SCENITH (Arguello et al. Cell Metabolism 2020) for single cell metabolic studies, we demonstrate that SB-4826 increases the BMR (Fig. 1B), mitochondrial dependency and proliferative capacity of tumor infiltrating PD1+ TIM3+ CD8+ T-cells (TILs). While both SB-4826 and PD1 inhibition led to increased CD8+ TIL numbers, SB-4826 led to increased BMR in CD8+ TILs while PD1 inhibition did not affect CD8+ TILs BMR. Additionally, PD1 inhibition significantly decreased CD8+ TILs mitochondrial dependency for metabolism, while SB-4826 increased mitochondrial dependency (Fig. 1B). Additionally, we recapitulate these metabolic enhancements in an in vitro system of T-cell activation, further confirming a T-cell intrinsic effect following SB-4826. Mechanistically, we found that SB-4826 led to increased HIF-1a levels in both CD4+ and CD8+ cells following TCR activation, likely through stabilization of HIF-1a due to decreased SUMOylation-dependent turnover (Cheng J et al. Cell 2007). We hypothesize that metabolic reprograming of CD8 T-cells through increased HIF-1a is the basis for synergy between SUMOylation inhibition and immune checkpoint inhibitors in LBCL.

Immunohistochemical Tumor Expression Predicts Response to CD3xCD20 Bispecific Antibodies in Patients with Relapsed/Refractory B Cell Lymphoma

Introduction Several studies show that CD3xCD20 bispecific antibodies show promising results in relapsed or refractory B-cell lymphomas and may have a milder adverse effect profile compared to chimeric antigen receptor T cell therapy (CAR-T). Tumor loss of antibody target (i.e. CD19 or PD1) have shown to negatively affect treatment outcomes in CD19-CAR-T cell therapy in DLBCL, and anti-PD1 therapy in Hodgkin lymphoma. It has, to our knowledge, not been reported how loss of CD20 expression and other immunohistochemical (IHC) expression patterns affects treatment outcomes in patients treated with CD3xCD20 antibodies. Methods We collected data on all patients in Denmark who received CD3xCD20 bispecific antibodies from 2017 to 2023. Electronic health records were assessed for general baseline characteristics and response according to Lugano criteria. Pathology reports on pre- and post-CD3xCD20 therapy lymphoma samples were collected and assessed on IHC analysis of CD20, Ki67, CD10, Bcl2, Bcl6, MUM1, germinal center B-cell (GCB) subtype and non-germinal center B-cell subtypes (non-GCB) according to Hans algorithm, as well as reports on cytogenetic FISH analysis regarding MYC with BCL2 and/or BCL6 rearrangements. To determine whether an expression was weak, negative, or strong the assessment of the original pathology reports was used. Results Pre-treatment IHC assessments were available in 122 patients. These were distributed across 81 patients with DLBCL, 32 with FL and 9 with other lymphoid neoplasms (MZL, HL, CLL). Of these, 109 had a strong CD20 expression, 10 had a weak CD20 expression, while 3 had no expression of CD20. Patients with a weak CD20 expression before treatment had an overall survival (OS) rate after 12 months at 40% (95% CI, 9.5-70) compared to 69% in patients with strong CD20 expression (95% CI, 45-61) (Figure 1, p=0.08). In a univariable Cox regression, we found that negative CD20 expression and higher Ki67 was significantly associated with inferior OS (Table 1, respectively HR=2.11 and HR=1.02 (per %-change in Ki67)). Worse performance status and a higher number of previous lines of treatment were also significantly associated with inferior overall survival (respectively HR=1.85 and HR=1.20). Surprisingly, BCL6 rearrangement was significantly associated with superior OS (HR=0.46). In a multivariable cox regression using OS as the endpoint, weak and negative IHC expression of CD20 was significantly associated with inferior OS (respectively HR=3.29, 95% CI 1.32-8.23, p=0.011 and HR=6.79 95% CI 1.25-36.76, p=0.026). Positive BCL6 compared to negative was associated with increased OS (HR =0.48, 95% CI 0.24 - 0.94, p=0.032). Analyzing only patients with DLBCL (n=81), weak and negative IHC expression of CD20 were the only factors to remain significantly associated with inferior overall survival (respectively HR=3.01, 95% CI 1.10-8.28, p=0.032 and HR=7.26, 95% CI 1.35-43.35, p=0.026). BCL6 rearrangement was associated with an increased overall survival, however only borderline statistically significant (HR=0.53, 95% CI 0.26-1.08, p=0.082). With regards to treatment response weak CD20 expression, worse performance status, higher Ki67 and a higher number of prior lines of treatment was in univariable analysis significantly associated with not being in a complete remission (CR) (respectively HR=2.28, HR=1.73, HR=1.01(per %-change in Ki67), HR=1.23 (per prior line of treatment). In a multivariable cox regression only weak and negative CD20 expression remained significantly associated with not being in CR (respectively HR=2.40, 95% CI 1.1-5.25, p=0.028 and HR=5.7 95% CI 1.14-28.58, p=0.034). Of the 48 patients who had biopsy-confirmed relapse, 29 had a weak or negative IHC CD20 expression (24 with a negative expression and 5 with a weak expression). Of these 29 patients, 24 had a strong pre-treatment IHC CD20 expression. Conclusions To our knowledge, we here present the first long-term analysis of the prognostic effect of tumor immunohistochemistry properties in patients treated with CD3xCD20 bispecific antibodies. We find that CD20 expression before treatment is an important prognostic factor, as patients with lymphomas with weak CD20 expression yield significantly poorer responses and decreased overall survival. Additionally, we find that BCL6 rearrangement is associated with superior overall survival in patients treated with CD3XCD20 bispecific antibodies.

Photoablation with the Aurolase System Reduces T Cell Exhaustion and Synergizes with Immunotherapies in Lymphoma

Introduction: Immunotherapy is less active in non-Hodgkin lymphoma due to immune evasion from the tumor microenvironment. For diffuse large B cell lymphoma (DLBCL), the overall response rates (ORR) for immune checkpoint inhibitors (ICIs) were low (4-18%). Bispecific antibodies (BiAb), such as FDA-approved Epcoritamab and Glofitamab, had a complete response (CR) rate of 39% for DLBCL. Methods that alter local and systemic immune microenvironments are needed to improve the response to immunotherapy. Unlike radiation, photothermal therapy (PTT) can generate a strong T-cell activation signal. PTT is created by irradiating specially designed gold nanoparticles with near-infrared (NIR) light. The nanoparticles absorb the light and transfer the energy into heat, leading to very localized ablations. The local high heat stimulates a cascade of pro-inflammatory cytokines, including IL-6, IL-1β, TNF-α, G-CSF, GM-CSF, and CCL2. We previously found that PTT with toll-like receptor 9 agonists had a significantly higher CD8 to CD4 ratio, cytotoxic T cell to regulatory T cell ratio, and effector memory T cells in a murine lymphoma model. The AuroLase system, which uses silica gold nanoshells and an FDA-cleared laser, has been undergoing clinical trials for various solid tumors. Here, we explore the systemic immune effects of the PTT device in a lymphoma murine model and evaluate its synergistic effects with ICIs and BiAbs. Methods: The AuroLase system and AuroShells were provided by Nanospectra. The lymphoma model uses A20 cells implanted in Balb/c mice. Single flank tumors were used for immunophenotyping, and a dual tumor model was used to evaluate the systemic anti-lymphoma effects of PTT and combination therapies. PTT (day 0) was performed by injecting 20ul of nanoshells intratumorally and then irradiating the tumor with the laser at 4W for 30sec-1min. Spleens were collected on days (d) 1, 4, and 8 for immunophenotype by flow cytometry. For systemic immune response, we used a dual tumor model and treated the left side with PTT on d0. Anti-PD1 (aPD1) antibodies and the CD20xCD3 BiAbs were injected intraperitoneally on d1, 3, and 5. Results: In a single tumor lymphoma model, PTT did not change the size of the spleen or the total T cell count, including CD4, CD8, and regulatory T cell (Treg), compared to tumor naïve or tumor-bearing mice. However, the treatment reduced splenocyte T cell PD1 levels on d1 &amp; 4 post-ablation, down to the level of tumor naive mice, and returned to elevated levels by d8, similar to untreated mice. CD4 and CD8 T cell PD1 levels dropped from 3.49% to 0.78% and 28% to 0.45%, respectively, post-ablation (p&amp;lt;0.001). Treg PD1 levels also dropped from 11.5% to 2.4%. There was a reduction of the central memory T cells (CD44 hi CD62 hi) population on d1&amp;4 post-ablation but started to increase by d8. The PD1 expression pattern was similar to the prior, with a reduction on d1&amp;4 and recovery by d8. The effector memory T cells (CD44 hi CD62 lo) had an increased population by d8 with the same PD1 reduction pattern. The B cell population didn't change, but there was an increase in B cell maturation (CD80 +) on d1&amp;4. As for the myeloid cells, there is a sharp increase in dendritic cell population on d1 and increased maturation/activation on d1&amp;4. There was an increased population of macrophages on d4. There was a reduction of myeloid-derived suppressor cells on d1. In the dual tumor model, the treated tumor had a dramatic loss of tumor volume due to PTT. The untreated tumor did not have a tumor reduction with PTT alone. However, when combined with anti PD1 (aPD1) therapy with just three doses, the untreated side had significant tumor growth reduction. By day 20, the PTT+aPD1 group had a tumor size of 378 mm 3, while the PTT alone and aPD1 alone groups were 1207 and 985 mm 3, respectively. There was also a survival advantage with PTT+aPD1. Similarly, we found that PTT+BiAb significantly delayed tumor growth compared to PTT only. Survival data is still ongoing. Conclusion: PTT in lymphoma is understudied and can effectively activate an anti-lymphoma response. PTT resulted in a marked reduction in PD1 expression on all T cells and activated several myeloid immune cells. We found that PTT combined with aPD1 therapy or BiAbs had improved tumor suppression in a lymphoma model. This system provides a platform and opportunity to translate PTT into the clinics.

Macrophages Play a Key Role in Controlling Tumor Growth and Response to Immunotherapy in Primary Central Nervous System Lymphoma

Introduction Primary Central Nervous System Lymphoma (PCNSL) represents 5% of extranodal lymphomas. Current treatments include high-dose chemotherapy in combination with an anti-CD20 antibody, and whole-brain radiation, both of which are limited by their toxicity. Although some advances have been achieved during the last few years, the prognosis of patients with CNS lymphoma is substantially worse than those with other type of lymphomas, with 5-year survival rates around 30%. The highly aggressive nature of CNS lymphomas is in part due to the fact that the brain is an immunoprivileged organ; it presents very low levels of immunosurveillance, which contributes to inefficient immune responses against malignant cells. Of note, up to 70% of patients diagnosed with PCNSL also exhibit genetic alterations to avoid recognition by the immune system, like loss of MHC-I, that prevent presentation of tumor antigens, and amplifications in PD-L1, which increase the negative stimuli in cytotoxic T cells. The PD-1/PD-L1 interaction has mainly been considered to be a checkpoint that regulates T cells. However, it has recently been found that tumor-associated macrophages can also express PD-1 and become activated to attack tumor cells when it is blocked. Moreover, macrophages have also been found to be inhibited by MHC-I expression on cancer cells. Thus, cancer cells that downregulate MHC-I to avoid T cell surveillance are particularly vulnerable to macrophages phagocytosis, especially when other macrophage immune checkpoints are blocked, such as CD47. Against this background, we aimed to decipher the differential role of macrophages and T cells in the tumor growth and response to immunotherapy in primary CNS lymphoma. Methods Luciferase-expressing A20 murine B-cell lymphoma cells were genetically modified to knock-out MHCI or MHCII by CRISPR-Cas9 and injected into the brain of immunocompetent (IC) or macrophage-depleted (MD) mice. Once tumours were stablished, mice were treated intravenously with seven injections, twice a week, of anti-PD1, anti-CD47 or the combination of both. We monitored mice for survival and tumoral growth. Additionally, brains were collected from mice treated with three injections for analysis of macrophages, B cells and T cells using flow cytometry and IHC. Results In IC mice, all anti-PD1 treated mice showed significant differences in tumoral growth and survival compared to vehicle; however, MHCI- tumors had the lowest overall survival (Figure 1A). With anti-CD47 therapy, only MHCI- PCNSL mice had a significantly increased survival compared to vehicle. Combination was effective in all groups, but no drug synergy was seen. In MD mice, all tumors were more aggressive than in IC mice. With anti-PD1, MHCI+ PCNSL mice achieved a complete tumor regression and survived longer in comparison to vehicle. On the contrary, MHCI- PCNSL mice didn't respond to anti-PD1 therapy (Figure 1B). Of notice, MHCI+ PCNSL in both MD and IC models had a higher infiltration of active T cells when treated with anti-PD1. In MHCI- PCNSL, infiltrated T cells had less expression of activation markers in both models and macrophages were polarized to an M2 phenotype in IC mice. Conclusions Our study revealed a remarkable response to anti-PD1 therapy in MHCI+ CNS lymphoma tumors, regardless of the presence of macrophages in the tumor microenvironment. This indicates that T cells would be primarily responsible for the observed tumour regression. However, targeting CD47 in MHCI+ PCNSL was found to be insufficient in restoring macrophage phagocytosis. Despite observing positive effects of anti-PD1 and anti-CD47 drugs in our MHCI- immunocompetent mouse model, MHCI downregulation still induces a very aggressive, lethal disease. This suggests that these tumors can only be controlled by macrophages, as supported by the lack of treatment response in a MD microenvironment. Our findings highlight the critical role of macrophages in controlling the growth of lymphoma cells in the brain and their significance in the immunotherapy response for CNS lymphomas, particularly when MHCI expression is deficient. Blocking PD1 exhibits superior efficacy when macrophages are present, indicating T cells are not the only player in this tumoral scenario. As a result, our study suggests that a promising approach for treating primary CNS lymphomas would involve targeting both T cells and macrophages.