PD-1 blockade does not improve efficacy of EpCAM-directed CAR T-cells in lung cancer brain metastasis.

PD-1 blockade does not improve efficacy of EpCAM-directed CAR T-cells in lung cancer brain metastasis. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article PD-1 blockade does not improve efficacy of EpCAM-directed CAR T-cells in lung cancer brain metastasis. Jens Blobner, Laura Dengler, Constantin Eberle, Julika J. Herold, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4456398/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Oct, 2024 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted 11 You are reading this latest preprint version Abstract Background Lung cancer brain metastasis have a devastating prognosis, necessitating innovative treatment strategies. While chimeric antigen receptor (CAR) T-cells show promise in hematologic malignancies, their efficacy in solid tumors, including brain metastasis, is limited by the immunosuppressive tumor environment. The PD-L1/PD-1 pathway inhibits CAR T-cell activity in the tumor microenvironment, presenting a potential target to enhance therapeutic efficacy. This study aims to evaluate the impact of anti-PD1 antibodies on CAR T-cells in treating lung cancer brain metastasis. Methods We utilized a murine immunocompetent, syngeneic orthotopic cerebral metastasis model for repetitive intracerebral two-photon laser scanning microscopy (TPLSM), enabling in vivo characterization of red fluorescent tumor cells and CAR T-cells at a single-cell level over time. Red fluorescent EpCAM-transduced Lewis Lung carcinoma cells ( EpCAM/tdt LL/2 cells) were implanted intracranially. Following the formation of brain metastasis, EpCAM-directed CAR T-cells were injected into adjacent brain tissue, and animals received either anti-PD-1 or an isotype control. Results Compared to controls receiving T-cells lacking a CAR, mice receiving EpCAM-directed CAR T-cells showed higher intratumoral CAR T-cell densities in the beginning after intraparenchymal injection. This finding was accompanied with reduced tumor growth and translated into a survival benefit. Additional anti-PD1 treatment, however, did not affect intratumoral CAR T-cell persistence nor tumor growth and thereby did not provide an additional therapeutic effect. Conclusion CAR T-cell therapy for brain malignancies appears promising. However, additional anti-PD1 treatment did not enhance intratumoral CAR T-cell persistence or effector function, highlighting the need for novel strategies to improve CAR T-cell therapy in solid tumors. CAR T cells brain metastasis lung cancer PD1-blockade Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Lung cancer remains a fatal disease for the great majority of patients.[ 1 ] Approximately, 10% of newly diagnosed patients present with brain metastasis and 25–40% develop brain metastases in the course of disease.[ 2 , 3 ] Conventional radiotherapy, chemotherapy, and novel treatment strategies including immune checkpoint inhibitors have improved the survival of non-small cell lung cancer (NSCLC) patients in the past decades, but the 5-year survival rate especially of patients with brain metastases remains poor.[ 4 ] Adoptive T-cell therapy with genetically modified T-cells that express synthetic receptors on the cell surface to detect and eradicate cancer cells by identifying specific tumor antigens, so called Chimeric antigen receptor (CAR) T-cells, has emerged as one of the most promising cancer immunotherapy modalities[ 5 ]. CAR T-cells targeting the B-cell lineage antigen CD19, demonstrate remarkable antitumor efficacy in the treatment of hematologic cancers. Six CAR T-cell therapies have been granted approval for hematological cancers by the Food and Drug Administration (FDA) since 2017.[ 6 ] Substantial efforts to improve the activity of CAR T-cells against solid tumors including tumors located in the CNS have been made, however their efficacy remains limited.[ 7 – 9 ] Currently, several clinical trials for CAR T-cells in the treatment of lung cancer are underway.[ 10 ] The epithelial cell adhesion molecule (EpCAM) may represent a target for CAR T-cell therapy as EpCAM is overexpressed in about 50% of non small cell lung cancer and their metastases.[ 11 – 14 ] According to our recent preclinical data, EpCAM-directed CAR T-cells may indeed constitute an effective therapeutic avenue for brain metastases from lung cancer. Additionally, and first clinical data shows that EpCAM-directed CAR T-cells are both safe and efficacious in the treatment of EpCAM-positive malignancies.[ 15 ] , [ 16 ] However, tumor escape from immune elimination is a hallmark of cancer.[ 17 ] Especially CNS tumors impair CAR T-cell efficacy by an adaptive immune suppressive response including upregulation of immune inhibitory molecules, such as programmed cell death ligand-1 (PD-L1) resulting in immune cell exhaustion.[ 18 ] This highlights the need for additional therapeutic strategies to counteract the hostile tumor microenvironment (TME) and overcome tumor heterogeneity. Immune Checkpoint blocking agents (ICB) can elicit durable clinical responses by reactivating an exhausted immune response and are part of standard the standard of care treatment in NSCLC.[ 19 ] Therefore, combining CAR T-cell therapy and ICB may produce a synergistic effects: Anti-PD-1 antibody therapy could restore the activity and functional persistence of not only the injected CAR T-cells but also other innate immune cells, including tumor-infiltrating lymphocytes, in the tumor microenvironment and may provide more efficient eradication of cancer cells.[ 20 , 21 ] In the current study, we combined a chronic cranial window with repetitive two photon laser scanning microscopy to establish an immunocompetent, syngeneic, orthotopic mouse model of lung cancer brain metastasis. This enables us to the repeatedly visualize fluorescent EpCAM-expressing Lewis Lung cancer cells and CAR T-cells at single cell level. Thereby, we can analyze in vivo dynamics and persistence and were eventually able to evaluate putative synergistic effects of an additional anti-PD-1 treatment. Methods Cell lines and cell culturing The LL/2 murine Lewis Lung carcinoma cell line was obtained from the European Collection of Authenticated Cell Cultures (ECACC; catalogue number: #90-0201-04). The cells were cultured in DMEM (Sigma-Aldrich; D6429) supplemented with 10% FBS (Sigma-Aldrich; F0804), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Sigma-Aldrich; P4333) at 37°C and 5% CO2. Regular testing was conducted to detect mycoplasma infection. The cells were maintained in culture for up to four weeks after thawing to minimize genetic drift. No evidence of Mycoplasma, squirrel monkey retrovirus, or interspecies contamination was found. Tumor cell line generation The generation of red fluorescent LL2/Epcam cells was carried out as previously described.[ 16 ] In brief, the lentiviral expression vector pLVX-IRES-neoR (LentiX-Bicistronic Expression System; catalog number #632181; TaKaRa Clontech) was utilized to clone a PCR product containing the coding sequence of tdTomato (vector ptdTomato; catalog number #63-2531; TaKaRa Clontech). The resulting construct pLVX-tdTomato-IRES-Neo, incorporating a resistance sequence for G418-sulfate, was validated through restriction enzyme digestion and Sanger sequencing.[ 22 ] Subsequently, LL/2 cells were transfected with pLVX-tdTomato-IRES-Neo using lipofection (Lipofectamine 3000; Thermo Fisher Scientific). Enrichment was achieved through cultivation in selection medium containing G418-sulfate and Geneticin (#A2912; Biochrom), along with repetitive FACS sorting. As previously described in detail[ 23 ], tdt LL/2 cells were stably transduced with a pMXs vector containing the full-length murine EpCAM (UNIPROT entry: #Q99JW5) cDNA, resulting in the creation of the EpCAM-overexpressing cell line EpCAM/tdt LL/2. Retroviral CAR vector In our study the anti-EpCAM CAR construct was used as previously described.[ 24 ] The murine EpCAM antigen (clone G8.8) is recognized by a single-chain variable fragment fused to the transmembrane and signaling domains of murine CD28 and murine CD3ζ in a pMP71 backbone. The anti-EpCAM-CAR-GFP construct comprises the anti-EpCAM-CAR fused to GFP through a self-cleaving 2A sequence. Transduction was conducted to generate GFP-fluorescent, EpCAM-directed CAR T-cells ( GFP/EpCAM CAR T-cells). For the generation of GFP T-cells, a pMP71 retroviral vector containing only eGFP was utilized. T-cell isolation and transduction CAR T-cells were generated as described previously.[ 16 ] Briefly, spleens of naïve C57BL6/j mice were harvested directly after cervical dislocation and a single-cell suspension was obtained using a 35 µm cell strainer (Greiner Bio-One; 542070). Erythrocytes underwent lysis utilizing ACK lysis buffer (comprising 150 mM NH4Cl, 10mM KHCO3, and 100 µM Na2EDTA) for a duration of 2 minutes. Following this, cells were rinsed with PBS. The transduction of primary murine T-cells was carried out in accordance with prior descriptions.[ 24 ] In brief, splenocytes were cultured in RPMI 1640 containing 10% fetal calf serum (FCS), 0.025% l-glutamine, 0.1% HEPES, 0.001% gentamicin, and 0.002% streptomycin supplemented with 25 U/mL IL-2; and stimulated overnight with anti-mouse CD3 antibodies (1:1,000, clone 145–2C11; BD Pharmingen) and anti-mouse CD28 antibodies (1:5,000, clone 37.51; BD Pharmingen). Supernatants containing the corresponding virus were used for the transduction procedure. During virus generation, T-cells were stimulated with Dynabeads Mouse T-Activator CD3/CD28 (#11-452D, Thermo Fisher). Transduced murine T-cells were cultured in murine T-cell medium enriched with recombinant human IL-7 (#200-007; Peprotech), IL-15 (#200 − 015; Peprotech), and β-mercaptoethanol (M6250-100; Sigma-Aldrich) for a maximum period of 7 days. Following retroviral transduction and expansion, EpCAM/GFP CAR T-cells and GFP T-cells were selected by CD3- and GFP-positivity utilizing a MoFlow Cell Sorter (Beckman Coulter) after exclusion of dead cells. Animals Male C57BL/6J wildtype mice were purchased from Charles River (Sulzfeld, Germany) at the age of 8–12 weeks and were kept under pathogen-controlled condition. Sex- and age-matched mice were used for further experiments. All animal experiments were approved by the local governmental animal care committee (permission number: 02-20-44) of the Ludwig-Maximilians-University Munich, Germany. The experiments were conducted in compliance with European legislation regarding animal protection and adhered to NIH Guidelines (NIH Publication #85 − 23 Rev. 1985). Surgical Procedure and postoperative care Preparation of a chronic cranial window was performed as previously described.[ 22 , 25 , 26 ], Briefly, a circular section of the skull (with a 5.5 mm diameter) was excised using a sterile carbon steel microdrill. Following this, the dura mater was separated from the below leptomeninges using two forceps in order to prevent dural fibrosis and optimize image resolution. Subsequently, the brain surface was coated with PBS, and a sterile round cover-glass was applied. For enhanced head positioning during imaging, a custom-made ring (made of polyether ether ketone [PEEK]) was securely affixed to the cranial bone using acrylic dental glue (Cyano veneer). Buprenorphine (0.1mg/kg; q8h) was administered for two consecutive days to ensure postoperative analgesia. To minimize postoperative microglia activation and preserve the integrity of intracranial microcirculation, a recovery period of 28 days was observed before proceeding with additional experiments. Intraparenchymal tumor cell inoculation and CAR T-cell injection After being resuspended in 1 µL PBS, stereotactically injection of 2.5x10 3 EpCAM/tdt LL/2 was performed into the left hemisphere at predefined coordinates (1 mm lateral to the sagittal sinus and 2 mm posterior to the bregma; intraparenchymal depth: 1.3 mm). Depending on the experimental setup, injection was done either after borehole placement (survival analysis) or careful removal of the chronic cranial window ( in vivo imaging analysis). At day 4 after tumor cell inoculation, mice were randomized to the different treatment groups. For immune checkpoint blockade 250 µg anti-PD-1 (RMP1-14, BioXCell) per mouse or equivalent doses of isotype control antibodies (2A3, BioXCell) were administered by intraperitoneal (i.p.) injection in 200 µl PBS every 3 days starting 4 days after tumor cell injection. The crossing of the blood-brain barrier and the intracerebral efficacy of the aPD-1 in vivo antibodies was shown recently in a murine brain tumor model[ 27 ]. Seven days after tumor cell injection 2x10 5 EpCAM/GFP CAR T-cells or GFP T-cells resuspended in 1 µL PBS were injected 1 mm posterior to the injection point (intraparenchymal depth: 1.3 mm) either after borehole placement (survival analysis) or careful removal of the chronic cranial window ( in vivo imaging analysis). Two Photon Laser Scanning Microscopy (TPLSM) TPLSM data were acquired using a Multiphoton TrimScope II system (LaVision BioTec) coupled with an upright Olympus microscope featuring a TiSA Coherent Chameleon Ultra-II-Femtolaser (wavelength 800 to 1,080 nm; Spectra Physics, Newport). The data was obtained using either a 4x objective (NA 0.28; Olympus XLFluor 4×/340) or a 20x water immersion objective (numerical aperture [NA] 0.95; Olympus XLUMPlanFl). During the imaging process, the mice were positioned on a heating mat and anesthetized with isoflurane in oxygen. The concentration of isoflurane was adjusted to 1.0 to 2.0% based on the breathing rate. To minimize movement during imaging, a PEEK ring was glued adjacent to the chronic cranial window and fixed using a custom-made fixation device. For enhanced visualization of intra- and extratumoral cerebral vessels, 0.1 ml of fluorescein isothiocyanate (FITC)-dextran (2 MDa molecular mass) was intravenously injected at a concentration of 10 mg/ml. Imaging starts at the surface (determined by detecting arachnoid fibers using second harmonic imaging) and progressed every 5 µm up to a depth of 400 µm. Image resolution was set at 1024 × 1024 pixels with a wavelength of 920nm. Three-dimensional (3D) stacks and dynamic images were captured using x/y/z-dimensions of 450 × 450 × 400 µm. Dynamic analyses involved 3D image stacks with x/y/z-dimensions of 450 × 450 × 66 µm. Images were acquired repetitively over 20 minutes, starting 100 µm below the cortical surface to ensure intratumoral imaging. Image Analysis ImageJ/Fiji and Imaris (Bitplane AG) were used for image processing and analysis. To ensure an unbiased processing approach, raters were kept blinded to group allocation until the final data analysis. To measure tumor size using in vivo microscopy, we quantified the number of fluorescent pixels of the 2D tumor area using epifluorescence microscopy. CAR T-cells were identified by their green fluorescent signal, while tumor cells were recognized by their red fluorescent signal. Immunohistochemical analysis Following cardiac perfusion, the brains were removed and fixed in 4% PFA. To facilitate dehydration, the samples underwent a series of sucrose incubations until equilibrium was reached. Subsequently, the brains were exposed to the gas phase of liquid nitrogen for 5 minutes before being stored at -80°C. Finally, the brains were sectioned into 15 µm-thick slices with a spacing of 495 µm. To quantify (CAR) T-cell counts, sections were stained with a chicken anti-GFP antibody (#AB13970; 1:200, Abcam) to specifically detect the GFP signal associated with (CAR) T-cells. The secondary antibody used was a goat anti-chicken AlexaFluor® 488 antibody (#AB150169; 1:200, Abcam). For investigations related to the immunosuppressive tumor microenvironment, sections underwent additional staining with a rabbit anti-Iba1 antibody (#01919741; 1:500, Wako) or rat anti-CD3 antibody (MAB4841, 15 µg/mL R&D). The applied secondary antibodies were chicken anti-rabbit AlexaFluor® 647 antibody (#A21443; 1:100, Invitrogen) and chicken anti-rat AlexaFluor® 647 antibody (#A21472; 1:100, Invitrogen), respectively. Following an overnight incubation in a humidified box at 4°C with the primary antibodies, secondary antibody labeling occurred at room temperature for 1 hour. The staining of cell nuclei was achieved using DAPI (#236276; 1:1000, Roche). The sections were analyzed using a Zeiss AxioImager M2 upright microscope from Carl Zeiss Microscopy. To calculate the tumor volume and CAR T-cell density through immunofluorescence, the tumor was manually outlined based on the fluorescence signal using the Zen Lite software package (version 2.3; Carl Zeiss Microscopy). The total tumor area per slice was then multiplied by a thickness of 495 µm, and the sum of these values yielded the overall tumor volume. The CAR T-cell density was calculated by dividing the total number of cells by the total tumor volume. Additionally, intratumoral CAR T-cells were categorized based on their location, either in the tumor core or the border zone (0–10 µm from the outer tumor edge). To analyze CD3 and Iba1, one to two random tumor slices per mouse were selected, and the density of CD3-positive cells was measured per tumor area. Detected cells were classified based on their location in either the tumor core or border zone. Flow cytometry and Fluorescence-Activated Cell Sorting (FACS) Flow cytometry and cell sorting were performed on a Beckman Coulter MoFlo Astrios cell sorter. To validate homogeneous expression of tdTomato, LL/2 tumor cells underwent flow cytometry analysis. During CAR T-cell production, EpCAM/GFP CAR T-cells and GFP T-cells were selected based on CD3- and GFP-positivity using a MoFlow Cell Sorter (Beckman Coulter) after retroviral transduction and expansion. Dead cells were excluded. Study protocol After the preparation of the cranial window, the mice were allowed to recover for a period of at least 21 days. To monitor tumor development using TPLSM, 2500 tumor cells were injected at standardized coordinates into the brain parenchyma. On day 4 after tumor cell inoculation, baseline epifluorescence imaging is conducted, and mice are then randomly assigned to one of the four groups: one receiving EpCAM/GFP CAR T-cells and anti-PD-1 (n = 8), one receiving GFP T-cells and anti-PD-1(n = 9), one receiving EpCAM/GFP CAR T-cells and IgG (n = 8) and one receiving GFP T-cells and IgG (n = 8). CAR T-cell injection was performed seven days after tumor cell injection. Intraperitoneal injection of anti-PD-1 and IgG, respectively, started 3 days prior to CAR T-cell administration and continued every 3 days until termination of the experiment (Fig. 1A). In vivo microscopy was performed on day 4, 7, 10, 13 and 16 after CAR T-cell injection. On day − 2 2D-epifluorescence imaging was done to confirm tumor take and for baseline tumor measurement. Animals were sacrificed by intracardiac injection of 0.9% NaCl solution followed by PFA 4% at the end of in vivo microscopy (day 16 after CAR T-cell injection) or when termination criteria were met. Brains were excised for immunofluorescence experiments. Burr hole injection of tumor cells and CAR T-cells, respectively, was done for survival experiments. On this occasion animals were sacrificed if termination criteria were met. Statistics Statistical analysis was performed using GraphPad Prism software (v9.0). Unless stated otherwise, normal distribution was assessed using the D’Agostino-Pearson test. Differences were evaluated using the Student's t-test for parametric data or the Mann-Whitney U-test for non-parametric data. Associations among categorical variables were assessed using the χ2-test. All data are presented as mean ± SEM. Absolute numbers and percentages are used to describe categorical variables. The Kaplan-Meier method was used for survival analysis, accompanied by the log-rank test. The significance threshold was set at p ≤ 0.05. The main manuscript contains all the necessary data to validate the findings of this study. Results Development of a robust murine model for repetitive imaging of lung cancer brain metastases Microsurgical implantation of a chronic cranial window was well tolerated, enabling repetitive in vivo imaging at identical coordinates through two-photon laser scanning microscopy. After inoculation of 2.5 x 10 3 EpCAM/tdt LL/2 tumor cells all mice (n = 8 per group) had visible tumors at day 5 after tumor cell injection (Fig. 1A). These cells form solitary lesions with a subsequent exponential growth pattern (Fig. 1B). The window quality and fluorescence of tumor cells and CAR T-cells was persistent, facilitating the visualization of the stereotactically implanted tumor cells as well as CAR T-cells at a single-cell resolution over several weeks. In vitro cytotoxicity of EpCAM-directed CAR T-cells. To ensure optimal in vivo functionality, physiological cytokine and chemokine production, proliferation, and migration patterns, we generated murine GFP-expressing EpCAM/GFP CAR T-cells using murine transmembrane and costimulatory domains. To evaluate the specific cytotoxicity of this newly generated EpCAM CAR we co-cultured FACS-sorted EpCAM-directed CAR T-cells with EpCAM/tdt LL/2 target cells. To control unspecific T-cell receptor (TCR)-mediated xenogeneic cytotoxicity of murine T-cells independent of CAR-mediated effects, we conducted a comparison of the killing activities of T-cells that were transduced with the same vector but lacked the CAR construct. After a 48-hour co-culturing period, only EpCAM-directed CAR T-cells show dose-depending cytotoxicity that was not detectable for sham-transduced T-cells illustrating the ability of EpCAM-directed CAR T-cells to specifically target and kill EpCAM/tdt LL/2 target cells without any bystander cells (Fig. 1C). In vivo behavior of CAR T-cells after intraparenchymal injection As previously demonstrated by our group intracranial but not intravenous injection of EpCAM/GFP CAR T-cells results in sufficient tumor control[ 16 ]. Therefore, we sought to analyze intracerebral CAR T-cell trafficking and antitumor efficacy after intraparenchymal injection. Seven days after tumor cell inoculation 2 x 10 5 EpCAM/GFP CAR T-cells were stereotactically administered into the brain parenchyma 1 mm adjacent to the tumor. To demonstrate CAR specificity, control mice were injected with GFP T-cells of similar numbers. Anti-PD-1 treatment was started 3 days before CAR T-cell injection to provide sufficient systemic drug levels. Quantifying intratumoral CAR T-cell density per TPLSM showed that both EpCAM/GFP CAR T-cells and GFP T-cells accumulate intratumorally over time on day 4 following injection. Intratumoral numbers of EpCAM/GFP CAR T-cells exceed those of GFP T-cells illustrating target tropism and successful tumor infiltration (Fig. 2A). Although a relevant number of EPCAM/GFP CAR T-cells were also found in the contralateral hemisphere, no significant differences in (CAR) T-cell densities were found compared to GFP T-cell-treated controls (Fig. 2C). This might indicate enhanced proliferation or migration of intratumoral EPCAM/GFP CAR T-cells rather than passive diffusion from the injection site alone. Effects of immune checkpoint blockade on the efficacy of CAR T-cell therapy Especially in solid tumors, adoptively transferred T-cells face an immunosuppressive microenvironment leading to T-cell exhaustion. Therefore, we aimed to elucidate whether repetitive intraperitoneal administration of PD-1-blocking antibodies may restore T-cell effector function, reduce tumor growth and prolong survival. Tumors in mice treated with GFP T-cells exhibited an exponential growth pattern, resulting in substantial tumor sizes by day 10 after CAR T-cell injection, with 87.5% (7/8) of the animals displaying tumor sizes exceeding 1 mm² (Fig. 3H). In contrast, mice treated with EpCAM/GFP CAR T-cells demonstrated a reduced growth rate, and none of the animals (0/8) reached a tumor size greater than 1 mm² (Fig. 3G). By day 10 following CAR T-cell injection, there was a reduction of tumor growth (0.32 mm² ± 0.26 vs. 5.44 mm² ± 7.04; p = 0.002) (Fig. 3A,D,G). This reduction was accompanied by the intratumoral accumulation of CAR T-cells (Fig. 2A + D). In one out of eight animals (12.5%), the injection of EpCAM/GFP CAR T-cells resulted in a complete regression of the tumor and no visible tumor was observed in the in vivo imaging from day 4 until the end of the experiment (Fig. 3G). Interestingly, additional anti-PD-1 treatment did not increase intratumoral EpCAM/GFP CAR T-cell density or anti-tumor efficacy, resulting in a similar growth pattern compared to animals receiving the IgG isotype control antibody (Fig. 2B + E and 3B-C + E-F). In a separate set of experiments focused on overall survival, burr hole trepanation was performed instead of cranial window implantation. As previously demonstrated, we observed a reduction in tumor growth after intraparenchymal injection of EpCAM/GFP CAR T-cells, which was accompanied by a survival benefit compared to GFP T-cells (Fig. 3I). It is noteworthy that concomitant anti-PD1 treatment was not able to ameliorate tumor-induced T-cell exhaustion, resulting in similar growth patterns and overall survival rates between the EpCAM/GFP CAR T-cell/anti-PD-1 and EpCAM/GFP CAR T-cell/IgG treated animals, respectively (Fig. 3B,E and J). In vivo CAR T-cell dynamics and spatial distribution below visualizable depths Repeated in vivo two-photon laser scanning microscopy provides reliable imaging of tumor-immune cell interactions down to a depth of 400 µm. To validate our 2-photon imaging findings and gain further insights into the anti-tumor effects and spatiotemporal distribution of locally injected CAR T-cells, we conducted immunofluorescence analyses of excised brains from animals treated with CAR T-cells, with and without simultaneous anti-PD1 treatment. The brains of mice treated with EpCAM/GFP CAR T-cells (± anti-PD1/IgG antibodies) and mice treated GFP T-cells (± anti-PD1/IgG antibodies) were collected between day 10 and day 16 following intracerebral CAR T-cell injection when mice met termination criteria. Tumors were found in 15 of 16 (93.8%) mice treated with EpCAM/GFP CAR T-cells (± anti-PD1/IgG antibodies) and in all mice of GFP T-cells (± anti-PD1/IgG antibodies). Immunofluorescence analysis of tumor volumes did not reveal differences between mice treated with EpCAM/GFP CAR T-cells and concurrent aPD-1 treatment and those given EpCAM/GFP CAR T-cells with the isotype control antibody (40 mm 3 ± 27.8 mm 3 vs. 61 mm 3 ± 19 mm 3 , p = 0.25) (Fig. 4E) further confirming our results of in vivo microscopy. Furthermore, we compared intratumoral density of EpCAM/GFP CAR T-cells in animals with and without concurrent aPD1 treatment. Consistent with our prior in vivo microscopy results, additional immune checkpoint blockade demonstrated no relevant impact on intratumoral infiltration by CAR T-cells (Fig. 4A-B). Furthermore, the spatial distribution within the tumor was found to be similar between both experimental groups. Notably, elevated quantities of CAR T-cells were detected within the tumor core in contrast to the tumor border (Fig. 4C). However, it should be noted that the absolute number of CAR T-cells at the end of the experiment was limited. Discussion Lung cancer is the leading cause of cancer deaths, with approximately 20% of patients diagnosed with metastatic disease.[ 28 ] Although therapeutic strategies for lung cancer brain metastases have advanced, CNS spread still significantly affects survival and quality of life.[ 29 ] While immune checkpoint inhibitors show promise in some cases, most patients do not respond to these therapies, emphasizing the need for new treatments.[ 30 ] Challenges in cellular-based approaches for solid brain tumors include the consistent identification of targets.[ 31 ] We used a fully immunocompetent murine model of lung cancer brain metastases and combined a chronic cranial window with repeated in vivo TPLSM to explore real-time dynamics of CAR T-cells at a single-cell level during combined administration of anti-PD1 and CAR T-cells. Our findings demonstrate the efficacy of EpCAM-directed CAR T-cells after intracerebral administration, resulting in a reduced tumor growth and prolonged survival. However, additional systemic anti-PD1 treatment did not increase the intratumoral persistence or the anti-tumor effects of CAR T-cells. Locoregional injection of CAR T-cells into the surrounding brain tissue not only resulted in a noteworthy reduction in tumor growth but also achieved complete regression in selected cases. Consequently, mice receiving EpCAM-directed CAR T-cells showed significantly prolonged survival rates compared to control animals. These outcomes substantiate the findings established in our prior investigations using this model and are in line with similar observations in different tumor entities, including medulloblastoma or ependymoma[ 16 ] , [ 32 ]. For T-cell suppression within the brain TME the PD-1/PD-L1 axis has been show to play a pivotal role.[ 33 , 34 ] Antigen contact induces CAR T-cell effector function and production of IFN-γ. Next, IFN-γ binds to its receptor initiating the JAK/STAT signaling pathway, which regulates PD-L1 expression on brain tumor cells and tumor associated macrophages.[ 35 ] Accordingly, first clinical data indicates that anti-EGFRVIII-CAR T-cell infusion can paradoxically promote immunosuppressive tumor microenvironment via upregulating inhibitory immune checkpoint molecules in glioblastoma.[ 36 ] Interestingly, a phase I clinical trial investigating repeated peripheral infusions of anti-EGFRvIII CAR T cells in combination with pembrolizumab was not effective in glioblastoma.[ 37 ] Several publications highlight the CNS penetrance and effectiveness of ICB antibodies in brain metastasis. Anti-PD1 treatment may reverse the immunosuppression within the TME and CNS tumors have been shown to respond to combined immune checkpoint blockade, resulting in elevated proportions of tumor-infiltrating lymphocytes (TILs).[ 27 , 38 , 39 ] Within the context of CAR T-cell treatment PD-1 suppression can be achieved through the co-administration of PD-1 targeting monoclonal antibodies or the PD-1 gene editing of CAR T-cells.[ 40 ] In systemic tumor models, the additional value of PD-1 blockade to increase CAR T-cell efficacy has been debated. In a murine preclinical model for systemic melanoma, concurrent PD-1 blockade notably increased the persistence and efficacy of CAR T-cell treatment.[ 41 ] However, in another study using a immunocompetent murine model for systemic melanoma, PD-1 blockade primarily mediates its anti-tumor effect through endogenous T-cells and did not increase the anti-tumor effect of CAR T-cell treatment.[ 42 ] Furthermore, it has been demonstrated, that PD-1 silencing may impair the anti-tumor function of CAR T-cells by inhibiting proliferation activity in a murine model of systemic NSCLC.[ 43 ] Song et al. demonstrated that anti-EGFRvIII CAR T-cell therapy with PD-1 checkpoint blockade in a CNS tumor model using U87 glioma cells.[ 44 ] However, it's noteworthy that these experiments were conducted in immunodeficient mice, which may overlook the influence of endogenous T-cell immunity and an intact PD-1–PD-L1 signaling axis. In our study, we used autologous spleenocytes for CAR T-cell production and observe no significant difference in intratumoral densities of CAR T-cells and CD3 + T-cells, nor in CAR T-cell persistence and survival following the co-administration of anti-PD-1 and CAR T-cells. Transduction with the retroviral CAR vector endows CAR T-cells with dual specificity via the CAR and the endogenous T-cell receptor (TCR). Although CAR T-cell-based therapies are recommended for the treatment of hematological malignancies, the effects of endogenous TCR signaling in CAR T-cell biology have not been well defined. Recent preclinical and clinical studies suggest that endogenous TCR signaling is not required for CAR T-cell effector function, whereas it could negatively affect proliferation and effector function[ 45 , 46 ]. Another potential mechanism contributing to the limited efficacy of the combinatorial approach is the complex composition of the tumor microenvironment (TME) within the brain which frequently harbors fewer proliferating immune cells compared to primary tumors and other metastatic sites. Additionally, T-cells in brain metastases exhibit elevated expression levels of immune checkpoint proteins compared to those in other sites, while macrophages in the brain are more prone to expressing an immune-suppressing M2 gene signature. These factors collectively contribute to impeding the effectiveness of CAR T-cells in CNS tumors.[ 47 – 49 ] By utilizing repetitive in vivo TPLSM we are capable of elucidating intratumoral CAR T-cell dynamics from early stages of tumor formation until late timepoints, when large tumors have formed. After intracerebral injection of CAR T-cells, we initially observed higher intratumoral densities of EpCAM-directed CAR T-cells compared to undirected CAR T-cells. In general, CAR T-cells recognize surface antigens independently from MHC restriction. Based on the intracranial administration, early contact of EpCAM-directed CAR T-cells with EpCAM-transuced LL/2 tumor cells may lead to receptor-antigen-interaction inducing activation, proliferation and the development of a cytotoxic phenotype.[ 50 , 51 ] Interestingly, intratumoral CAR T-cell density and proliferation diminished during the observation period indicating insufficient CAR T-cell persistence within the tumor. Consequently, a decreasing amount of EpCAM-directed CAR T-cells was paralleled by tumor growth. Consistent with our data, several preclinical and clinical studies in other solid brain tumors observe decreasing CAR T-cell numbers and T-cell exhaustion even when a sufficient T-cell infiltration has been achieved.[ 52 , 53 ] Immunologically, large tumor burden requires persistent CAR T-cell function upon repeated antigen stimulation in an immunosuppressive environment to eventually achieve tumor eradication. However, chronic antigenic stimulation by the tumor results in endogenous T-cell exhaustion characterized by loss of lytic function and cytokine secretion with simultaneous expression of inhibitory receptors like PD1/PD-L1[ 54 , 55 ]. Consequently, we sought to elucidate the impact of concomitant anti-PD1 treatment on CAR T-cell migration and effector function. Surprisingly, we do not observe any differences in CAR T-cell migration to and persistence within the tumor after anti-PD1 treatment. In line with that, no survival differences could be observed between animals receiving ICB and the isotype control antibodies, respectively. In general, anti-PD-1 antibodies mainly function by disrupting the interaction between PD-1 on T-cells and PD-L1 on tumor cells. Paucity of PD-L1 on tumor cells is a well-defined factor associated with resistance to anti-PD-1 antibody treatment while high expression usually indicate better response rates.[ 56 – 58 ] However, the PD-L1 expression varies among patients and between different tumor entities.[ 59 ] Furthermore, the upregulation of alternative immune checkpoints or the activation of alternative signaling pathways within tumor cells may contribute to resistance. For instance, tumor cells may exploit pathways other than the PD-1-PD-L1 axis to evade immune surveillance. Liu et al. engineered CAR T-cells by modifying PD-1, incorporating the extracellular and transmembrane domains of PD-1 with the intracellular signaling domain of CD28. This adaptation facilitated the transformation of inhibitory signals within the TME into activating signals.[ 60 ] The resultant 'switch-receptor' CAR T-cells exhibited enhanced efficacy in tumor control compared to the concurrent administration of anti-PD-1 with CAR T-cells. The conversion of multiple inhibitory signals within the TME to stimulatory signals holds significant potential for improving anti-tumor cytotoxicity. This is particularly noteworthy given the elevated expression of checkpoints, including PD-1, LAG-3, TIM-3, and TIGIT, along with their ligands, in solid brain tumors.[ 61 ] Converting the ubiquitous inhibitory signals into stimulatory signals can thereby greatly improve CAR T-cell infiltration and persistence and has to be investigated in further studies. Although CAR T-cell therapy shows promising results in B cell malignancies, CNS affection is a common exclusion criterion in clinical trials mainly driven by fear of neurotoxicity. Additionally, most CARs targeting solid tumors use antigens shared by normal tissues, carrying the risk of on-target off-tumor toxicity. The additional use of ICB theoretically increases efficacy while also increasing the risk of toxicity. Such side effects most frequently comprise neurological symptoms, epileptic seizures, systemic immune reactions like the cytokine release syndrome (CRS), organ dysfunction and death[ 62 , 63 ]. Amongst others, the CAR construct in our model is constituted by an scFv capable of recognizing murine EpCAM in most epithelial tissues. As a pan-epithelial marker, EpCAM is homogenously expressed on the surface of healthy alveolar tissue[ 64 ]. Due to shared expression on the surface of tumor cells and healthy tissue, the risk of on-target-off-tumor reactions is significantly increased in the context auf EpCAM-directed CAR T-cells and anti-PD1 treatment[ 65 ]. Notably, we did not observe any clinically relevant side-effects in our fully immunocompetent mouse model. Nevertheless, it remains to be mentioned that especially due to the small sample size and the translational nature of our experimental set-up we cannot fully predict on on-target/off-tumor reactions of our combinatorial approach. In conclusion, we demonstrated that locally injected CAR T-cells adjacent to the tumor lead to intratumoral accumulation and reduced tumor growth translating into a survival benefit of EpCAM/GFP CAR T-cell treated mice. Even though additional anti-PD1 treatment was safe and well-tolerated, it does not elicit unconstrained proliferation or intratumoral persistence. Declarations Funding J.B. acknowledges research grants from the Munich Clinician Scientist Program “Else-Kröner-Fresenius Forschungskolleg” and of the Medical Faculty of the Ludwig-Maximilians-University Munich. T.X. acknowledge scholarship support from China Scholarship Council (CSC). KJM acknowledges research grants from the Friedrich-Baur-Foundation, from the “Society for Research and Science at the Medical Faculty of the LMU” at the Ludwig-Maximilians-University Munich, and from the SFB TRR 338 (project B02). N.T. acknowledges a research grant from the "Support Program for Research and Teaching" at the Ludwig-Maximilians-University Munich. H.I-A acknowledges the SFB 914 (project Z01). P.K. acknowledges research grants from the Friedrich-Baur-Foundation, from the "Support Program for Research and Teaching" at the Ludwig-Maximilians-University Munich, from the “Society for Research and Science at the Medical Faculty of the LMU” at the Ludwig-Maximilians-University Munich, and from the “Familie Mehdorn”-Foundation. L.v.B. acknowledges support by the SFB TRR 338 (project B02) and from the advanced Munich Clinical Scientist Program” and the Helene and Bruno Jöster foundation. This study was supported by the Bavarian Cancer Research Center (BZKF) (TANGO to S.K.), the Deutsche Forschungsgemeinschaft (DFG, KO5055-2-1 and KO5055/3-1 to S.K.), the international doctoral program ‘i-Target: immunotargeting of cancer’ (funded by the Elite Network of Bavaria; to S. K.), the Melanoma Research Alliance (grant number 409510 to S.K.), Marie Sklodowska-Curie Training Network for Optimizing Adoptive T Cell Therapy of Cancer (funded by the Horizon 2020 program of the European Union; grant 955575 to S.K.), Else Kröner-Fresenius-Stiftung (IOLIN to S.K.), German Cancer Aid (AvantCAR.de to S.K.), the Wilhelm-Sander-Stiftung (to S. K.), Ernst Jung Stiftung (to S.K.), Institutional Strategy LMUexcellent of LMU Munich (within the framework of the German Excellence Initiative; to S.K.), the Go-Bio-Initiative (to S.K.), the m4-Award of the Bavarian Ministry for Economic Affairs (to S.K.), Bundesministerium für Bildung und Forschung (to S.K.), European Research Council (Starting Grant 756017, PoC Grant 101100460 and CoG 101124203 to S.K.), by the SFB-TRR 338/1 2021–452881907 (to S.K.), Fritz-Bender Foundation (to S.K.), Deutsche José Carreras Leukämie Stiftung (to S.K.), Hector Foundation (to S.K.), Bavarian Research Foundation (BAYCELLATOR to S.K.), the Bruno and Helene Jöster Foundation (360° CAR to S.K.), the Monika-Kutzner Stiftung (to S.K.). Conflict of Interest Disclosures Jens Blobner - No disclosures Laura Dengler - No disclosures Constantin Eberle - No disclosures Julika Herold - No disclosures Tao Xu - No disclosures Alexander Beck - No disclosures Anton Mühlbauer - No disclosures Katharina Mueller - No disclosures Philipp Karschnia - No disclosures Nico Teske - No disclosures Dominic van den Heuvel - No disclosures Ferdinand Schallerer - No disclosures Hellen Ishikawa-Ankerhold - No disclosures Marion Subklewe - No disclosures Niklas Thon - No disclosures Jörg-Christian Tonn - Research grants from Novocure and Munich Surgical Imaging; and Royalties from Springer Publisher Intl. Marion Subklewe - No disclosures Sebastian Kobold - S. K. has received honoraria from TCR2 Inc., Miltenyi, Galapagos, Novartis, BMS and GSK. S. K. is an inventor of several patents in the field of immuno-oncology. S. K. received license fees from TCR2 Inc and Carina Biotech. S.K. received research support from TCR2 Inc., Tabby Therapeutics, Catalym GmBH, Plectonic GmBH and Arcus Bioscience for work unrelated to the manuscript. Patrick N. Harter - No disclosures Veit R Buchholz - No disclosures Louisa von Baumgarten - No disclosures Author Contributions Statement J.B., L.v.B. conceptualization, planning and drafting of the manuscript J.B., L.D. performed the main experiments. J.B., L.D., C.E., J.J.H., A.B., A.M. D.v.H., F.S data acquisition and analysis. K.J.M., N.Te, P.K. helped in interpreting the data. All authors reviewed the manuscript. References Ramalingam SS, Owonikoko TK, Khuri FR (2011) Lung cancer: New biological insights and recent therapeutic advances. CA Cancer J Clin 61:91–112. https://doi.org/10.3322/caac.20102 Barnholtz-Sloan JS, Sloan AE, Davis FG et al (2004) Incidence proportions of brain metastases in patients diagnosed (1973 to 2001) in the Metropolitan Detroit Cancer Surveillance System. 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OncoImmunology 9:1806009. https://doi.org/10.1080/2162402X.2020.1806009 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure.tiff Supplementary Figure (A,B) Kaplan-Meier survival estimates for mice bearing brain tumors (injected seven days prior to local (CAR) T-cell administration), subsequent to treatment with either EpCAM/GFP CAR T-cells + aPD1 (dark green; n=8) and GFP CAR T-cells + aPD1 (dark gray; n=8), respectively (A) GFP T-cells + IgG isotype antibody (light gray, n=8) and GFP CAR T-cells + aPD1 (dark gray; n=8), respectively (B). Log-rank test (**p=0.0005). (C) Intratumoral (CAR) T-cell density (cells/mm 3 ) on d4, d7 and d10 after local injection of GFP T-cells (+ IgG isotyp (light gray; n=8)) and GFP T-cells + anti-PD1 antibodies (dark grey, n=9)), respectively as determined by two-photon laser scanning microscopy. (D) Summarized tumor areas (mm 2 ) of n=8 animals receiving locally injected GFP T-cells + IgG (light grey) and GFP T-cells + aPD1 (n=9, dark grey). Mean ± SEM. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4456398","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":309213868,"identity":"623cb3fe-33e6-4a88-8321-5e7c1a5ecd0d","order_by":0,"name":"Jens Blobner","email":"data:image/png;base64,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","orcid":"","institution":"Department of Neurosurgery, LMU University Hospital, Ludwig Maximilians University (LMU), 81377 Munich","correspondingAuthor":true,"prefix":"","firstName":"Jens","middleName":"","lastName":"Blobner","suffix":""},{"id":309213869,"identity":"9e4955f8-243f-462b-91ac-133f30d8b667","order_by":1,"name":"Laura Dengler","email":"","orcid":"","institution":"German Cancer Consortium (DKTK), Partner Site Munich, a partnership between the DKFZ Heidelberg and the University Hospital of the LMU","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Dengler","suffix":""},{"id":309213870,"identity":"1d0d8cfc-c9d4-4e79-80b6-90af1e79e6c1","order_by":2,"name":"Constantin Eberle","email":"","orcid":"","institution":"German Cancer Consortium (DKTK), Partner Site Munich, a partnership between the DKFZ Heidelberg and the University Hospital of the LMU","correspondingAuthor":false,"prefix":"","firstName":"Constantin","middleName":"","lastName":"Eberle","suffix":""},{"id":309213871,"identity":"80804080-f848-408d-96a7-96b18cf127b2","order_by":3,"name":"Julika J. Herold","email":"","orcid":"","institution":"German Cancer Consortium (DKTK), Partner Site Munich, a partnership between the DKFZ Heidelberg and the University Hospital of the LMU","correspondingAuthor":false,"prefix":"","firstName":"Julika","middleName":"J.","lastName":"Herold","suffix":""},{"id":309213872,"identity":"62317c4a-2ea4-4f83-a297-9e925bafd202","order_by":4,"name":"Tao Xu","email":"","orcid":"","institution":"Department of Neurology, LMU University Hospital, Ludwig Maximilians University (LMU), 81377 Munich","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Xu","suffix":""},{"id":309213873,"identity":"128cddea-1363-4642-b2c1-f961efe24e03","order_by":5,"name":"Alexander Beck","email":"","orcid":"","institution":"Center for Neuropathology and Prion Research, LMU University Hospital, Ludwig Maximilians University (LMU), 81377 Munich","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"","lastName":"Beck","suffix":""},{"id":309213874,"identity":"9b2de501-1bad-442b-82b5-0a72ef4cc9b8","order_by":6,"name":"Anton Muehlbauer","email":"","orcid":"","institution":"Institute for Medical Microbiology, Immunology and Hygiene, Technical University of Munich, 81675 Munich","correspondingAuthor":false,"prefix":"","firstName":"Anton","middleName":"","lastName":"Muehlbauer","suffix":""},{"id":309213875,"identity":"ce36388d-b61c-4954-9ef2-1938a59c169c","order_by":7,"name":"Katharina J. Müller","email":"","orcid":"","institution":"Department of Neurology, LMU University Hospital, Ludwig Maximilians University (LMU), 81377 Munich","correspondingAuthor":false,"prefix":"","firstName":"Katharina","middleName":"J.","lastName":"Müller","suffix":""},{"id":309213876,"identity":"6d2f7eb0-2d50-48c6-8086-fad12bb0564b","order_by":8,"name":"Nico Teske","email":"","orcid":"","institution":"Department of Neurosurgery, LMU University Hospital, Ludwig Maximilians University (LMU), 81377 Munich","correspondingAuthor":false,"prefix":"","firstName":"Nico","middleName":"","lastName":"Teske","suffix":""},{"id":309213877,"identity":"f5b6c2cc-c20d-4831-bf77-18c038aad987","order_by":9,"name":"Philipp Karschnia","email":"","orcid":"","institution":"Department of Neurosurgery, LMU University Hospital, Ludwig Maximilians University (LMU), 81377 Munich","correspondingAuthor":false,"prefix":"","firstName":"Philipp","middleName":"","lastName":"Karschnia","suffix":""},{"id":309213878,"identity":"9cb5e8cc-5f54-4269-843c-b7426e451304","order_by":10,"name":"Dominic van den Heuvel","email":"","orcid":"","institution":"Department of Medicine I, Ludwig-Maximilians-University School of Medicine, 81377 Munich","correspondingAuthor":false,"prefix":"","firstName":"Dominic","middleName":"van den","lastName":"Heuvel","suffix":""},{"id":309213879,"identity":"ddae383d-9ed9-4bac-ae12-e1059f516f24","order_by":11,"name":"Ferdinand Schallerer","email":"","orcid":"","institution":"German Cancer Consortium (DKTK), Partner Site Munich, a partnership between the DKFZ Heidelberg and the University Hospital of the LMU","correspondingAuthor":false,"prefix":"","firstName":"Ferdinand","middleName":"","lastName":"Schallerer","suffix":""},{"id":309213880,"identity":"a2239666-51aa-4421-932f-9cbadd2323ab","order_by":12,"name":"Hellen Ishikawa-Ankerhold","email":"","orcid":"","institution":"Department of Medicine I, Ludwig-Maximilians-University School of Medicine, 81377 Munich","correspondingAuthor":false,"prefix":"","firstName":"Hellen","middleName":"","lastName":"Ishikawa-Ankerhold","suffix":""},{"id":309213881,"identity":"894a6542-bab7-4942-91e0-a3676f734955","order_by":13,"name":"Niklas Thon","email":"","orcid":"","institution":"Department of Neurosurgery, LMU University Hospital, Ludwig Maximilians University (LMU), 81377 Munich","correspondingAuthor":false,"prefix":"","firstName":"Niklas","middleName":"","lastName":"Thon","suffix":""},{"id":309213882,"identity":"0240f3cc-6e7d-4d80-bd5d-a687b0dc7913","order_by":14,"name":"Joerg-Christian Tonn","email":"","orcid":"","institution":"Department of Neurosurgery, LMU University Hospital, Ludwig Maximilians University (LMU), 81377 Munich","correspondingAuthor":false,"prefix":"","firstName":"Joerg-Christian","middleName":"","lastName":"Tonn","suffix":""},{"id":309213883,"identity":"a51aa924-0a4a-4e8c-a1a2-0eb3303991c6","order_by":15,"name":"Marion Subklewe","email":"","orcid":"","institution":"Department of Medicine III, Ludwig-Maximilians-University School of Medicine, 81377 Munich","correspondingAuthor":false,"prefix":"","firstName":"Marion","middleName":"","lastName":"Subklewe","suffix":""},{"id":309213884,"identity":"7741eed7-adb0-4e66-a8e7-956ed5ffd8f5","order_by":16,"name":"Sebastian Kobold","email":"","orcid":"","institution":"Department of Medicine IV, Division of Clinical Pharmacology, LMU University Hospital Munich","correspondingAuthor":false,"prefix":"","firstName":"Sebastian","middleName":"","lastName":"Kobold","suffix":""},{"id":309213885,"identity":"4a53ab12-122d-4ca7-abbb-dc4d5390f3e4","order_by":17,"name":"Patrick N. Harter","email":"","orcid":"","institution":"Center for Neuropathology and Prion Research, LMU University Hospital, Ludwig Maximilians University (LMU), 81377 Munich","correspondingAuthor":false,"prefix":"","firstName":"Patrick","middleName":"N.","lastName":"Harter","suffix":""},{"id":309213886,"identity":"b4c03654-3b95-4962-aefe-69e46ecbacf6","order_by":18,"name":"Veit R. Buchholz","email":"","orcid":"","institution":"Institute for Medical Microbiology, Immunology and Hygiene, Technical University of Munich, 81675 Munich","correspondingAuthor":false,"prefix":"","firstName":"Veit","middleName":"R.","lastName":"Buchholz","suffix":""},{"id":309213887,"identity":"28055c67-45a5-4f11-a696-873ec41d3ff6","order_by":19,"name":"Louisa von Baumgarten","email":"","orcid":"","institution":"Department of Neurosurgery, LMU University Hospital, Ludwig Maximilians University (LMU), 81377 Munich","correspondingAuthor":false,"prefix":"","firstName":"Louisa","middleName":"","lastName":"von Baumgarten","suffix":""}],"badges":[],"createdAt":"2024-05-21 17:27:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4456398/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4456398/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00262-024-03837-9","type":"published","date":"2024-10-03T15:58:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57633163,"identity":"b89dae2c-6797-499e-8dba-83ab61514454","added_by":"auto","created_at":"2024-06-03 15:21:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":690625,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental protocol. (A) \u003c/strong\u003eSchematic illustrating the experimental setup, including chronology of tumor cell injection, CAR T-cell injection, ICB therapy, and imaging methodologies. \u003cstrong\u003e(B) \u003c/strong\u003eIntracerebral tumor growth following stereotactic implantation of \u003csup\u003eEpCAM/tdT\u003c/sup\u003eLL/2 tumor cells (red). Blood vessels are highlighted after i.v. injection of FITC-dextran (green). Images represent mosaics of multiple maximum intensity projections with 400\u0026nbsp;µm depth from the brain surface (Scale bar 200µm left pictures, 1000µm right picture). Note that the day count refers to the day after tumor cell injection \u003cstrong\u003e(C) \u003c/strong\u003eFlow cytometry of \u003csup\u003eEpCAM/tdT\u003c/sup\u003eLL/2 tumor cells after 48h of co-culturing with \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cells and \u003csup\u003eGFP\u003c/sup\u003eT-cells respectively.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4456398/v1/62020c05673cda9dd512c614.png"},{"id":57633167,"identity":"28cac086-c117-4fbc-827b-55e580bee6a2","added_by":"auto","created_at":"2024-06-03 15:21:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":111895,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTPLSM of \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eEpCAM/GFP\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eCAR T-cells and \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eGFP\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT-cells after intraparenchymal injection 7 days after tumor cell inoculation. (A, B) \u003c/strong\u003eIntratumoral (CAR) T-cell density (cells/mm\u003csup\u003e3\u003c/sup\u003e) on d4, d7 and d10 after local injection of \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cells (+ IgG isotyp (light green; n=8) or anti-PD1 antibodies (dark green, n=8)) and \u003csup\u003eGFP\u003c/sup\u003eT-cells (+ IgG isotyp (n=7)) respectively as determined by two-photon laser scanning microscopy.\u003cstrong\u003e (C)\u003c/strong\u003e Comparison of (CAR) T-cell densities (cells/mm\u003csup\u003e3\u003c/sup\u003e) on day 4 after injection within the tumor and the contralateral, non tumor-bearing hemisphere as assessed by TPLSM in maximum intensity projection (MIP). All results are displayed as Mean ± SEM. \u003cstrong\u003e(D, E)\u003c/strong\u003e Representative images of EpCAM-directed CAR T- (n=8) and undirected T-cells (n=7) with isotype controle IgG (D) or in combination with intraperitoneal anti-PD1-treatment (E) on d4, d7 and d10 after intraparenchymal administration using \u003cem\u003ein vivo\u003c/em\u003e TPLSM in MIP with 400µm depth from the brain surface. Scale bars 100µm. Mean ± SEM.\u003c/p\u003e","description":"","filename":"OnlineFigure22.png","url":"https://assets-eu.researchsquare.com/files/rs-4456398/v1/389b91c4afba8ceec909c6fe.png"},{"id":57633168,"identity":"2f7dc6e7-07a4-410b-8631-36301669f770","added_by":"auto","created_at":"2024-06-03 15:21:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":144843,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTumor growth and survival after intraparenchymal injection of (CAR) T-cells with and without concomitant aPD1 treatment. (A, B, C)\u003c/strong\u003e Summarized tumor areas (mm\u003csup\u003e2\u003c/sup\u003e) of n=8 animals receiving locally injected \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cells + IgG (A, B, light green) / + aPD1 (B,\u0026nbsp;dark green) and n=8 animals receiving \u003csup\u003eGFP\u003c/sup\u003eT-cells + IgG (A, C, light grey) / + aPD1 (C,\u0026nbsp;dark grey), respectively. Growth behavior was determined by \u003cem\u003ein vivo \u003c/em\u003emicroscopy using epifluorescence. Mean ± SEM. ***p ≤\u0026nbsp;0.0005. \u003cstrong\u003e(D,\u0026nbsp;E,\u0026nbsp;F) \u003c/strong\u003eBrain tumor growth illustrated by one representative animal on day -2, 4, 7 and 10 after local (CAR) T-cell administration and intraperitoneal anti-PD1/IgG isotype injection measured by TPLSM using epifluorescence. Tumor cells are visualized by their red fluorescent signal. Scale bars 400µm. \u003cstrong\u003e(G, H)\u003c/strong\u003e Individual tumor areas (mm\u003csup\u003e2\u003c/sup\u003e) of n=8 \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cells + IgG (G) and n=8 \u003csup\u003eGFP\u003c/sup\u003eT-cells + IgG (H), respectively, measured by \u003cem\u003ein vivo\u003c/em\u003e TPLSM using epifluorescence on days −2, 4, 7 and 10 after local injection.\u003cstrong\u003e (I,\u0026nbsp;J) \u003c/strong\u003eKaplan-Meier survival estimates for tumor bearing mice (injected seven days prior to local (CAR) T-cell administration) treated with either \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR\u0026nbsp;T-cells + aPD1 (dark green; n=8), \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR\u0026nbsp;T-cells\u0026nbsp;+\u0026nbsp;IgG\u0026nbsp;isotype antibody (light green; n=8) or \u003csup\u003eGFP\u003c/sup\u003eT-cells + IgG isotype antibody (light gray, n=8). Log-rank test, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4456398/v1/4f60f300cdbff358eb4606c6.png"},{"id":57633165,"identity":"ccd4e53f-41a8-4dc0-a4ae-29aa7bcbb004","added_by":"auto","created_at":"2024-06-03 15:21:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":576483,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of intratumoral CAR T-cell dynamics below visible depths using immunofluorescence. \u003c/strong\u003eAfter termination of the experiment brains get excised and stained for (CAR) T-cell density\u003cstrong\u003e. (A) \u003c/strong\u003eHistological sections of brains after tumor cell and \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cell injection and intraperitoneal aPD1 and IgG administration, respectively. Sections were stained with an antibody against GFP to identify CAR T-cells (green), against CD3 to visualize T-cells (pink) and DAPI for cell nuclei (blue). CAR T-cell density within the tumor and at the infiltration zone was analyzed. Please note: Tumor size do not differ significantly between both groups. Scale bars 1400µm (top images) and 150µm (small images). \u003cstrong\u003e(B) \u003c/strong\u003eIntratumoral CAR T-cell density (cells/mm\u003csup\u003e3\u003c/sup\u003e) at the end of the experiment with (n=4) and without (n=4) concomitant aPD1 treatment. \u003cstrong\u003e(C, D)\u003c/strong\u003e Percentage distribution of CAR T-cells within the tumor and at the infiltration zone determined by immunofluorescence. \u003cstrong\u003e(E)\u003c/strong\u003e Tumor volume (mm\u003csup\u003e3\u003c/sup\u003e) of \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cell treated animals with and without concomitant anti-PD1 treatment. Mean ± SEM.\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4456398/v1/5df0d0704fadc81ad84865bd.png"},{"id":66097055,"identity":"49ac64f4-d09f-40a6-ba05-421b208eb136","added_by":"auto","created_at":"2024-10-07 16:13:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2760111,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4456398/v1/7d47d10b-876d-4620-ab2d-297a7d0aaa79.pdf"},{"id":57633166,"identity":"a0d8bbeb-24b7-4505-9ad9-f59ac09aa929","added_by":"auto","created_at":"2024-06-03 15:21:37","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":247368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure (A,B) \u003c/strong\u003eKaplan-Meier survival estimates for mice bearing brain tumors (injected seven days prior to local (CAR) T-cell administration), subsequent to treatment with either \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR\u0026nbsp;T-cells + aPD1 (dark green; n=8) and \u003csup\u003eGFP\u003c/sup\u003eCAR\u0026nbsp;T-cells\u0026nbsp;+\u0026nbsp;aPD1 (dark gray; n=8), respectively (A) \u003csup\u003eGFP\u003c/sup\u003eT-cells + IgG isotype antibody (light gray, n=8) and \u003csup\u003eGFP\u003c/sup\u003eCAR\u0026nbsp;T-cells\u0026nbsp;+\u0026nbsp;aPD1 (dark gray; n=8), respectively (B). Log-rank test (**p=0.0005). (\u003cstrong\u003eC) \u003c/strong\u003eIntratumoral (CAR) T-cell density (cells/mm\u003csup\u003e3\u003c/sup\u003e) on d4, d7 and d10 after local injection of \u003csup\u003e\u0026nbsp;GFP\u003c/sup\u003eT-cells (+ IgG isotyp (light gray; n=8)) and \u003csup\u003eGFP\u003c/sup\u003eT-cells + anti-PD1 antibodies (dark grey, n=9)), respectively as determined by two-photon laser scanning microscopy. \u003cstrong\u003e(D) \u003c/strong\u003eSummarized tumor areas (mm\u003csup\u003e2\u003c/sup\u003e) of n=8 animals receiving locally injected \u003csup\u003eGFP\u003c/sup\u003eT-cells + IgG (light grey) and \u003csup\u003eGFP\u003c/sup\u003eT-cells + aPD1 (n=9, dark grey). Mean ± SEM.\u003c/p\u003e","description":"","filename":"SupplementaryFigure.tiff","url":"https://assets-eu.researchsquare.com/files/rs-4456398/v1/cf5c68467572ed65130c18c6.tiff"}],"financialInterests":"No competing interests reported.","formattedTitle":"PD-1 blockade does not improve efficacy of EpCAM-directed CAR T-cells in lung cancer brain metastasis.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLung cancer remains a fatal disease for the great majority of patients.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] Approximately, 10% of newly diagnosed patients present with brain metastasis and 25\u0026ndash;40% develop brain metastases in the course of disease.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] Conventional radiotherapy, chemotherapy, and novel treatment strategies including immune checkpoint inhibitors have improved the survival of non-small cell lung cancer (NSCLC) patients in the past decades, but the 5-year survival rate especially of patients with brain metastases remains poor.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eAdoptive T-cell therapy with genetically modified T-cells that express synthetic receptors on the cell surface to detect and eradicate cancer cells by identifying specific tumor antigens, so called Chimeric antigen receptor (CAR) T-cells, has emerged as one of the most promising cancer immunotherapy modalities[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. CAR T-cells targeting the B-cell lineage antigen CD19, demonstrate remarkable antitumor efficacy in the treatment of hematologic cancers. Six CAR T-cell therapies have been granted approval for hematological cancers by the Food and Drug Administration (FDA) since 2017.[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] Substantial efforts to improve the activity of CAR T-cells against solid tumors including tumors located in the CNS have been made, however their efficacy remains limited.[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eCurrently, several clinical trials for CAR T-cells in the treatment of lung cancer are underway.[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] The epithelial cell adhesion molecule (EpCAM) may represent a target for CAR T-cell therapy as EpCAM is overexpressed in about 50% of non small cell lung cancer and their metastases.[\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] According to our recent preclinical data, EpCAM-directed CAR T-cells may indeed constitute an effective therapeutic avenue for brain metastases from lung cancer. Additionally, and first clinical data shows that EpCAM-directed CAR T-cells are both safe and efficacious in the treatment of EpCAM-positive malignancies.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] However, tumor escape from immune elimination is a hallmark of cancer.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] Especially CNS tumors impair CAR T-cell efficacy by an adaptive immune suppressive response including upregulation of immune inhibitory molecules, such as programmed cell death ligand-1 (PD-L1) resulting in immune cell exhaustion.[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] This highlights the need for additional therapeutic strategies to counteract the hostile tumor microenvironment (TME) and overcome tumor heterogeneity.\u003c/p\u003e \u003cp\u003eImmune Checkpoint blocking agents (ICB) can elicit durable clinical responses by reactivating an exhausted immune response and are part of standard the standard of care treatment in NSCLC.[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] Therefore, combining CAR T-cell therapy and ICB may produce a synergistic effects: Anti-PD-1 antibody therapy could restore the activity and functional persistence of not only the injected CAR T-cells but also other innate immune cells, including tumor-infiltrating lymphocytes, in the tumor microenvironment and may provide more efficient eradication of cancer cells.[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIn the current study, we combined a chronic cranial window with repetitive two photon laser scanning microscopy to establish an immunocompetent, syngeneic, orthotopic mouse model of lung cancer brain metastasis. This enables us to the repeatedly visualize fluorescent EpCAM-expressing Lewis Lung cancer cells and CAR T-cells at single cell level. Thereby, we can analyze in vivo dynamics and persistence and were eventually able to evaluate putative synergistic effects of an additional anti-PD-1 treatment.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and cell culturing\u003c/h2\u003e \u003cp\u003eThe LL/2 murine Lewis Lung carcinoma cell line was obtained from the European Collection of Authenticated Cell Cultures (ECACC; catalogue number: #90-0201-04). The cells were cultured in DMEM (Sigma-Aldrich; D6429) supplemented with 10% FBS (Sigma-Aldrich; F0804), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Sigma-Aldrich; P4333) at 37\u0026deg;C and 5% CO2. Regular testing was conducted to detect mycoplasma infection. The cells were maintained in culture for up to four weeks after thawing to minimize genetic drift. No evidence of Mycoplasma, squirrel monkey retrovirus, or interspecies contamination was found.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTumor cell line generation\u003c/h2\u003e \u003cp\u003eThe generation of red fluorescent LL2/Epcam cells was carried out as previously described.[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] In brief, the lentiviral expression vector pLVX-IRES-neoR (LentiX-Bicistronic Expression System; catalog number #632181; TaKaRa Clontech) was utilized to clone a PCR product containing the coding sequence of tdTomato (vector ptdTomato; catalog number #63-2531; TaKaRa Clontech). The resulting construct pLVX-tdTomato-IRES-Neo, incorporating a resistance sequence for G418-sulfate, was validated through restriction enzyme digestion and Sanger sequencing.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] Subsequently, LL/2 cells were transfected with pLVX-tdTomato-IRES-Neo using lipofection (Lipofectamine 3000; Thermo Fisher Scientific). Enrichment was achieved through cultivation in selection medium containing G418-sulfate and Geneticin (#A2912; Biochrom), along with repetitive FACS sorting. As previously described in detail[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], \u003csup\u003etdt\u003c/sup\u003eLL/2 cells were stably transduced with a pMXs vector containing the full-length murine EpCAM (UNIPROT entry: #Q99JW5) cDNA, resulting in the creation of the EpCAM-overexpressing cell line \u003csup\u003eEpCAM/tdt\u003c/sup\u003eLL/2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRetroviral CAR vector\u003c/h2\u003e \u003cp\u003eIn our study the anti-EpCAM CAR construct was used as previously described.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] The murine EpCAM antigen (clone G8.8) is recognized by a single-chain variable fragment fused to the transmembrane and signaling domains of murine CD28 and murine CD3ζ in a pMP71 backbone. The anti-EpCAM-CAR-GFP construct comprises the anti-EpCAM-CAR fused to GFP through a self-cleaving 2A sequence. Transduction was conducted to generate GFP-fluorescent, EpCAM-directed CAR T-cells (\u003csup\u003eGFP/EpCAM\u003c/sup\u003eCAR T-cells). For the generation of \u003csup\u003eGFP\u003c/sup\u003eT-cells, a pMP71 retroviral vector containing only eGFP was utilized.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eT-cell isolation and transduction\u003c/h2\u003e \u003cp\u003eCAR T-cells were generated as described previously.[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] Briefly, spleens of na\u0026iuml;ve C57BL6/j mice were harvested directly after cervical dislocation and a single-cell suspension was obtained using a 35 \u0026micro;m cell strainer (Greiner Bio-One; 542070). Erythrocytes underwent lysis utilizing ACK lysis buffer (comprising 150 mM NH4Cl, 10mM KHCO3, and 100 \u0026micro;M Na2EDTA) for a duration of 2 minutes. Following this, cells were rinsed with PBS. The transduction of primary murine T-cells was carried out in accordance with prior descriptions.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] In brief, splenocytes were cultured in RPMI 1640 containing 10% fetal calf serum (FCS), 0.025% l-glutamine, 0.1% HEPES, 0.001% gentamicin, and 0.002% streptomycin supplemented with 25 U/mL IL-2; and stimulated overnight with anti-mouse CD3 antibodies (1:1,000, clone 145\u0026ndash;2C11; BD Pharmingen) and anti-mouse CD28 antibodies (1:5,000, clone 37.51; BD Pharmingen). Supernatants containing the corresponding virus were used for the transduction procedure. During virus generation, T-cells were stimulated with Dynabeads Mouse T-Activator CD3/CD28 (#11-452D, Thermo Fisher). Transduced murine T-cells were cultured in murine T-cell medium enriched with recombinant human IL-7 (#200-007; Peprotech), IL-15 (#200\u0026thinsp;\u0026minus;\u0026thinsp;015; Peprotech), and β-mercaptoethanol (M6250-100; Sigma-Aldrich) for a maximum period of 7 days. Following retroviral transduction and expansion, \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cells and \u003csup\u003eGFP\u003c/sup\u003eT-cells were selected by CD3- and GFP-positivity utilizing a MoFlow Cell Sorter (Beckman Coulter) after exclusion of dead cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eMale C57BL/6J wildtype mice were purchased from Charles River (Sulzfeld, Germany) at the age of 8\u0026ndash;12 weeks and were kept under pathogen-controlled condition. Sex- and age-matched mice were used for further experiments. All animal experiments were approved by the local governmental animal care committee (permission number: 02-20-44) of the Ludwig-Maximilians-University Munich, Germany. The experiments were conducted in compliance with European legislation regarding animal protection and adhered to NIH Guidelines (NIH Publication #85\u0026thinsp;\u0026minus;\u0026thinsp;23 Rev. 1985).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSurgical Procedure and postoperative care\u003c/h2\u003e \u003cp\u003ePreparation of a chronic cranial window was performed as previously described.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], Briefly, a circular section of the skull (with a 5.5 mm diameter) was excised using a sterile carbon steel microdrill. Following this, the dura mater was separated from the below leptomeninges using two forceps in order to prevent dural fibrosis and optimize image resolution. Subsequently, the brain surface was coated with PBS, and a sterile round cover-glass was applied. For enhanced head positioning during imaging, a custom-made ring (made of polyether ether ketone [PEEK]) was securely affixed to the cranial bone using acrylic dental glue (Cyano veneer). Buprenorphine (0.1mg/kg; q8h) was administered for two consecutive days to ensure postoperative analgesia. To minimize postoperative microglia activation and preserve the integrity of intracranial microcirculation, a recovery period of 28 days was observed before proceeding with additional experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eIntraparenchymal tumor cell inoculation and CAR T-cell injection\u003c/h2\u003e \u003cp\u003eAfter being resuspended in 1 \u0026micro;L PBS, stereotactically injection of 2.5x10\u003csup\u003e3 EpCAM/tdt\u003c/sup\u003eLL/2 was performed into the left hemisphere at predefined coordinates (1 mm lateral to the sagittal sinus and 2 mm posterior to the bregma; intraparenchymal depth: 1.3 mm). Depending on the experimental setup, injection was done either after borehole placement (survival analysis) or careful removal of the chronic cranial window (\u003cem\u003ein vivo\u003c/em\u003e imaging analysis).\u003c/p\u003e \u003cp\u003eAt day 4 after tumor cell inoculation, mice were randomized to the different treatment groups. For immune checkpoint blockade 250 \u0026micro;g anti-PD-1 (RMP1-14, BioXCell) per mouse or equivalent doses of isotype control antibodies (2A3, BioXCell) were administered by intraperitoneal (i.p.) injection in 200 \u0026micro;l PBS every 3 days starting 4 days after tumor cell injection. The crossing of the blood-brain barrier and the intracerebral efficacy of the aPD-1 in vivo antibodies was shown recently in a murine brain tumor model[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Seven days after tumor cell injection 2x10\u003csup\u003e5 EpCAM/GFP\u003c/sup\u003eCAR T-cells or \u003csup\u003eGFP\u003c/sup\u003eT-cells resuspended in 1 \u0026micro;L PBS were injected 1 mm posterior to the injection point (intraparenchymal depth: 1.3 mm) either after borehole placement (survival analysis) or careful removal of the chronic cranial window (\u003cem\u003ein vivo\u003c/em\u003e imaging analysis).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eTwo Photon Laser Scanning Microscopy (TPLSM)\u003c/h2\u003e \u003cp\u003eTPLSM data were acquired using a Multiphoton TrimScope II system (LaVision BioTec) coupled with an upright Olympus microscope featuring a TiSA Coherent Chameleon Ultra-II-Femtolaser (wavelength 800 to 1,080 nm; Spectra Physics, Newport). The data was obtained using either a 4x objective (NA 0.28; Olympus XLFluor 4\u0026times;/340) or a 20x water immersion objective (numerical aperture [NA] 0.95; Olympus XLUMPlanFl). During the imaging process, the mice were positioned on a heating mat and anesthetized with isoflurane in oxygen. The concentration of isoflurane was adjusted to 1.0 to 2.0% based on the breathing rate.\u003c/p\u003e \u003cp\u003eTo minimize movement during imaging, a PEEK ring was glued adjacent to the chronic cranial window and fixed using a custom-made fixation device. For enhanced visualization of intra- and extratumoral cerebral vessels, 0.1 ml of fluorescein isothiocyanate (FITC)-dextran (2 MDa molecular mass) was intravenously injected at a concentration of 10 mg/ml. Imaging starts at the surface (determined by detecting arachnoid fibers using second harmonic imaging) and progressed every 5 \u0026micro;m up to a depth of 400 \u0026micro;m. Image resolution was set at 1024 \u0026times; 1024 pixels with a wavelength of 920nm.\u003c/p\u003e \u003cp\u003eThree-dimensional (3D) stacks and dynamic images were captured using x/y/z-dimensions of 450 \u0026times; 450 \u0026times; 400 \u0026micro;m. Dynamic analyses involved 3D image stacks with x/y/z-dimensions of 450 \u0026times; 450 \u0026times; 66 \u0026micro;m. Images were acquired repetitively over 20 minutes, starting 100 \u0026micro;m below the cortical surface to ensure intratumoral imaging.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImage Analysis\u003c/h2\u003e \u003cp\u003eImageJ/Fiji and Imaris (Bitplane AG) were used for image processing and analysis. To ensure an unbiased processing approach, raters were kept blinded to group allocation until the final data analysis. To measure tumor size using \u003cem\u003ein vivo\u003c/em\u003e microscopy, we quantified the number of fluorescent pixels of the 2D tumor area using epifluorescence microscopy. CAR T-cells were identified by their green fluorescent signal, while tumor cells were recognized by their red fluorescent signal.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical analysis\u003c/h2\u003e \u003cp\u003eFollowing cardiac perfusion, the brains were removed and fixed in 4% PFA. To facilitate dehydration, the samples underwent a series of sucrose incubations until equilibrium was reached. Subsequently, the brains were exposed to the gas phase of liquid nitrogen for 5 minutes before being stored at -80\u0026deg;C. Finally, the brains were sectioned into 15 \u0026micro;m-thick slices with a spacing of 495 \u0026micro;m.\u003c/p\u003e \u003cp\u003eTo quantify (CAR) T-cell counts, sections were stained with a chicken anti-GFP antibody (#AB13970; 1:200, Abcam) to specifically detect the GFP signal associated with (CAR) T-cells. The secondary antibody used was a goat anti-chicken AlexaFluor\u0026reg; 488 antibody (#AB150169; 1:200, Abcam). For investigations related to the immunosuppressive tumor microenvironment, sections underwent additional staining with a rabbit anti-Iba1 antibody (#01919741; 1:500, Wako) or rat anti-CD3 antibody (MAB4841, 15 \u0026micro;g/mL R\u0026amp;D). The applied secondary antibodies were chicken anti-rabbit AlexaFluor\u0026reg; 647 antibody (#A21443; 1:100, Invitrogen) and chicken anti-rat AlexaFluor\u0026reg; 647 antibody (#A21472; 1:100, Invitrogen), respectively. Following an overnight incubation in a humidified box at 4\u0026deg;C with the primary antibodies, secondary antibody labeling occurred at room temperature for 1 hour. The staining of cell nuclei was achieved using DAPI (#236276; 1:1000, Roche).\u003c/p\u003e \u003cp\u003eThe sections were analyzed using a Zeiss AxioImager M2 upright microscope from Carl Zeiss Microscopy. To calculate the tumor volume and CAR T-cell density through immunofluorescence, the tumor was manually outlined based on the fluorescence signal using the Zen Lite software package (version 2.3; Carl Zeiss Microscopy). The total tumor area per slice was then multiplied by a thickness of 495 \u0026micro;m, and the sum of these values yielded the overall tumor volume. The CAR T-cell density was calculated by dividing the total number of cells by the total tumor volume. Additionally, intratumoral CAR T-cells were categorized based on their location, either in the tumor core or the border zone (0\u0026ndash;10 \u0026micro;m from the outer tumor edge). To analyze CD3 and Iba1, one to two random tumor slices per mouse were selected, and the density of CD3-positive cells was measured per tumor area. Detected cells were classified based on their location in either the tumor core or border zone.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry and Fluorescence-Activated Cell Sorting (FACS)\u003c/h2\u003e \u003cp\u003eFlow cytometry and cell sorting were performed on a Beckman Coulter MoFlo Astrios cell sorter. To validate homogeneous expression of tdTomato, LL/2 tumor cells underwent flow cytometry analysis. During CAR T-cell production, \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cells and \u003csup\u003eGFP\u003c/sup\u003eT-cells were selected based on CD3- and GFP-positivity using a MoFlow Cell Sorter (Beckman Coulter) after retroviral transduction and expansion. Dead cells were excluded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStudy protocol\u003c/h2\u003e \u003cp\u003eAfter the preparation of the cranial window, the mice were allowed to recover for a period of at least 21 days. To monitor tumor development using TPLSM, 2500 tumor cells were injected at standardized coordinates into the brain parenchyma. On day 4 after tumor cell inoculation, baseline epifluorescence imaging is conducted, and mice are then randomly assigned to one of the four groups: one receiving \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cells and anti-PD-1 (n\u0026thinsp;=\u0026thinsp;8), one receiving \u003csup\u003eGFP\u003c/sup\u003eT-cells and anti-PD-1(n\u0026thinsp;=\u0026thinsp;9), one receiving \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cells and IgG (n\u0026thinsp;=\u0026thinsp;8) and one receiving \u003csup\u003eGFP\u003c/sup\u003eT-cells and IgG (n\u0026thinsp;=\u0026thinsp;8). CAR T-cell injection was performed seven days after tumor cell injection. Intraperitoneal injection of anti-PD-1 and IgG, respectively, started 3 days prior to CAR T-cell administration and continued every 3 days until termination of the experiment (Fig.\u0026nbsp;1A). \u003cem\u003eIn vivo\u003c/em\u003e microscopy was performed on day 4, 7, 10, 13 and 16 after CAR T-cell injection. On day \u0026minus;\u0026thinsp;2 2D-epifluorescence imaging was done to confirm tumor take and for baseline tumor measurement. Animals were sacrificed by intracardiac injection of 0.9% NaCl solution followed by PFA 4% at the end of \u003cem\u003ein vivo\u003c/em\u003e microscopy (day 16 after CAR T-cell injection) or when termination criteria were met. Brains were excised for immunofluorescence experiments. Burr hole injection of tumor cells and CAR T-cells, respectively, was done for survival experiments. On this occasion animals were sacrificed if termination criteria were met.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPad Prism software (v9.0). Unless stated otherwise, normal distribution was assessed using the D\u0026rsquo;Agostino-Pearson test. Differences were evaluated using the Student's t-test for parametric data or the Mann-Whitney U-test for non-parametric data. Associations among categorical variables were assessed using the χ2-test. All data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Absolute numbers and percentages are used to describe categorical variables. The Kaplan-Meier method was used for survival analysis, accompanied by the log-rank test. The significance threshold was set at p\u0026thinsp;\u0026le;\u0026thinsp;0.05. The main manuscript contains all the necessary data to validate the findings of this study.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDevelopment of a robust murine model for repetitive imaging of lung cancer brain metastases\u003c/h2\u003e \u003cp\u003eMicrosurgical implantation of a chronic cranial window was well tolerated, enabling repetitive \u003cem\u003ein vivo\u003c/em\u003e imaging at identical coordinates through two-photon laser scanning microscopy. After inoculation of 2.5 x 10\u003csup\u003e3 EpCAM/tdt\u003c/sup\u003eLL/2 tumor cells all mice (n\u0026thinsp;=\u0026thinsp;8 per group) had visible tumors at day 5 after tumor cell injection (Fig.\u0026nbsp;1A). These cells form solitary lesions with a subsequent exponential growth pattern (Fig.\u0026nbsp;1B). The window quality and fluorescence of tumor cells and CAR T-cells was persistent, facilitating the visualization of the stereotactically implanted tumor cells as well as CAR T-cells at a single-cell resolution over several weeks.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro cytotoxicity of EpCAM-directed CAR T-cells.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo ensure optimal \u003cem\u003ein vivo\u003c/em\u003e functionality, physiological cytokine and chemokine production, proliferation, and migration patterns, we generated murine GFP-expressing \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cells using murine transmembrane and costimulatory domains. To evaluate the specific cytotoxicity of this newly generated EpCAM CAR we co-cultured FACS-sorted EpCAM-directed CAR T-cells with \u003csup\u003eEpCAM/tdt\u003c/sup\u003eLL/2 target cells. To control unspecific T-cell receptor (TCR)-mediated xenogeneic cytotoxicity of murine T-cells independent of CAR-mediated effects, we conducted a comparison of the killing activities of T-cells that were transduced with the same vector but lacked the CAR construct. After a 48-hour co-culturing period, only EpCAM-directed CAR T-cells show dose-depending cytotoxicity that was not detectable for sham-transduced T-cells illustrating the ability of EpCAM-directed CAR T-cells to specifically target and kill \u003csup\u003eEpCAM/tdt\u003c/sup\u003eLL/2 target cells without any bystander cells (Fig.\u0026nbsp;1C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo behavior of CAR T-cells after intraparenchymal injection\u003c/h2\u003e \u003cp\u003eAs previously demonstrated by our group intracranial but not intravenous injection of \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cells results in sufficient tumor control[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Therefore, we sought to analyze intracerebral CAR T-cell trafficking and antitumor efficacy after intraparenchymal injection. Seven days after tumor cell inoculation 2 x 10\u003csup\u003e5 EpCAM/GFP\u003c/sup\u003eCAR T-cells were stereotactically administered into the brain parenchyma 1 mm adjacent to the tumor. To demonstrate CAR specificity, control mice were injected with \u003csup\u003eGFP\u003c/sup\u003eT-cells of similar numbers. Anti-PD-1 treatment was started 3 days before CAR T-cell injection to provide sufficient systemic drug levels. Quantifying intratumoral CAR T-cell density per TPLSM showed that both \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cells and \u003csup\u003eGFP\u003c/sup\u003eT-cells accumulate intratumorally over time on day 4 following injection. Intratumoral numbers of \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cells exceed those of \u003csup\u003eGFP\u003c/sup\u003eT-cells illustrating target tropism and successful tumor infiltration (Fig.\u0026nbsp;2A). Although a relevant number of \u003csup\u003eEPCAM/GFP\u003c/sup\u003eCAR T-cells were also found in the contralateral hemisphere, no significant differences in (CAR) T-cell densities were found compared to \u003csup\u003eGFP\u003c/sup\u003eT-cell-treated controls (Fig.\u0026nbsp;2C). This might indicate enhanced proliferation or migration of intratumoral \u003csup\u003eEPCAM/GFP\u003c/sup\u003eCAR T-cells rather than passive diffusion from the injection site alone.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEffects of immune checkpoint blockade on the efficacy of CAR T-cell therapy\u003c/h2\u003e \u003cp\u003eEspecially in solid tumors, adoptively transferred T-cells face an immunosuppressive microenvironment leading to T-cell exhaustion. Therefore, we aimed to elucidate whether repetitive intraperitoneal administration of PD-1-blocking antibodies may restore T-cell effector function, reduce tumor growth and prolong survival. Tumors in mice treated with \u003csup\u003eGFP\u003c/sup\u003eT-cells exhibited an exponential growth pattern, resulting in substantial tumor sizes by day 10 after CAR T-cell injection, with 87.5% (7/8) of the animals displaying tumor sizes exceeding 1 mm\u0026sup2; (Fig.\u0026nbsp;3H). In contrast, mice treated with \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cells demonstrated a reduced growth rate, and none of the animals (0/8) reached a tumor size greater than 1 mm\u0026sup2; (Fig.\u0026nbsp;3G). By day 10 following CAR T-cell injection, there was a reduction of tumor growth (0.32 mm\u0026sup2; \u0026plusmn; 0.26 vs. 5.44 mm\u0026sup2; \u0026plusmn; 7.04; p\u0026thinsp;=\u0026thinsp;0.002) (Fig.\u0026nbsp;3A,D,G). This reduction was accompanied by the intratumoral accumulation of CAR T-cells (Fig.\u0026nbsp;2A\u0026thinsp;+\u0026thinsp;D). In one out of eight animals (12.5%), the injection of \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cells resulted in a complete regression of the tumor and no visible tumor was observed in the \u003cem\u003ein vivo\u003c/em\u003e imaging from day 4 until the end of the experiment (Fig.\u0026nbsp;3G). Interestingly, additional anti-PD-1 treatment did not increase intratumoral \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cell density or anti-tumor efficacy, resulting in a similar growth pattern compared to animals receiving the IgG isotype control antibody (Fig.\u0026nbsp;2B\u0026thinsp;+\u0026thinsp;E and 3B-C\u0026thinsp;+\u0026thinsp;E-F). In a separate set of experiments focused on overall survival, burr hole trepanation was performed instead of cranial window implantation. As previously demonstrated, we observed a reduction in tumor growth after intraparenchymal injection of \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cells, which was accompanied by a survival benefit compared to \u003csup\u003eGFP\u003c/sup\u003eT-cells (Fig.\u0026nbsp;3I). It is noteworthy that concomitant anti-PD1 treatment was not able to ameliorate tumor-induced T-cell exhaustion, resulting in similar growth patterns and overall survival rates between the \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cell/anti-PD-1 and \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cell/IgG treated animals, respectively (Fig.\u0026nbsp;3B,E and J).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo CAR T-cell dynamics and spatial distribution below visualizable depths\u003c/h2\u003e \u003cp\u003eRepeated \u003cem\u003ein vivo\u003c/em\u003e two-photon laser scanning microscopy provides reliable imaging of tumor-immune cell interactions down to a depth of 400 \u0026micro;m. To validate our 2-photon imaging findings and gain further insights into the anti-tumor effects and spatiotemporal distribution of locally injected CAR T-cells, we conducted immunofluorescence analyses of excised brains from animals treated with CAR T-cells, with and without simultaneous anti-PD1 treatment. The brains of mice treated with \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cells (\u0026plusmn;\u0026thinsp;anti-PD1/IgG antibodies) and mice treated \u003csup\u003eGFP\u003c/sup\u003eT-cells (\u0026plusmn;\u0026thinsp;anti-PD1/IgG antibodies) were collected between day 10 and day 16 following intracerebral CAR T-cell injection when mice met termination criteria. Tumors were found in 15 of 16 (93.8%) mice treated with \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cells (\u0026plusmn;\u0026thinsp;anti-PD1/IgG antibodies) and in all mice of \u003csup\u003eGFP\u003c/sup\u003eT-cells (\u0026plusmn;\u0026thinsp;anti-PD1/IgG antibodies). Immunofluorescence analysis of tumor volumes did not reveal differences between mice treated with \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cells and concurrent aPD-1 treatment and those given \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cells with the isotype control antibody (40 mm\u003csup\u003e3\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;27.8 mm\u003csup\u003e3\u003c/sup\u003e vs. 61 mm\u003csup\u003e3\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;19 mm\u003csup\u003e3\u003c/sup\u003e, p\u0026thinsp;=\u0026thinsp;0.25) (Fig.\u0026nbsp;4E) further confirming our results of \u003cem\u003ein vivo\u003c/em\u003e microscopy. Furthermore, we compared intratumoral density of \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cells in animals with and without concurrent aPD1 treatment. Consistent with our prior \u003cem\u003ein vivo\u003c/em\u003e microscopy results, additional immune checkpoint blockade demonstrated no relevant impact on intratumoral infiltration by CAR T-cells (Fig.\u0026nbsp;4A-B). Furthermore, the spatial distribution within the tumor was found to be similar between both experimental groups. Notably, elevated quantities of CAR T-cells were detected within the tumor core in contrast to the tumor border (Fig.\u0026nbsp;4C). However, it should be noted that the absolute number of CAR T-cells at the end of the experiment was limited.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eLung cancer is the leading cause of cancer deaths, with approximately 20% of patients diagnosed with metastatic disease.[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] Although therapeutic strategies for lung cancer brain metastases have advanced, CNS spread still significantly affects survival and quality of life.[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] While immune checkpoint inhibitors show promise in some cases, most patients do not respond to these therapies, emphasizing the need for new treatments.[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] Challenges in cellular-based approaches for solid brain tumors include the consistent identification of targets.[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eWe used a fully immunocompetent murine model of lung cancer brain metastases and combined a chronic cranial window with repeated \u003cem\u003ein vivo\u003c/em\u003e TPLSM to explore real-time dynamics of CAR T-cells at a single-cell level during combined administration of anti-PD1 and CAR T-cells. Our findings demonstrate the efficacy of EpCAM-directed CAR T-cells after intracerebral administration, resulting in a reduced tumor growth and prolonged survival. However, additional systemic anti-PD1 treatment did not increase the intratumoral persistence or the anti-tumor effects of CAR T-cells.\u003c/p\u003e \u003cp\u003eLocoregional injection of CAR T-cells into the surrounding brain tissue not only resulted in a noteworthy reduction in tumor growth but also achieved complete regression in selected cases. Consequently, mice receiving EpCAM-directed CAR T-cells showed significantly prolonged survival rates compared to control animals. These outcomes substantiate the findings established in our prior investigations using this model and are in line with similar observations in different tumor entities, including medulloblastoma or ependymoma[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor T-cell suppression within the brain TME the PD-1/PD-L1 axis has been show to play a pivotal role.[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] Antigen contact induces CAR T-cell effector function and production of IFN-γ. Next, IFN-γ binds to its receptor initiating the JAK/STAT signaling pathway, which regulates PD-L1 expression on brain tumor cells and tumor associated macrophages.[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] Accordingly, first clinical data indicates that anti-EGFRVIII-CAR T-cell infusion can paradoxically promote immunosuppressive tumor microenvironment via upregulating inhibitory immune checkpoint molecules in glioblastoma.[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] Interestingly, a phase I clinical trial investigating repeated peripheral infusions of anti-EGFRvIII CAR T cells in combination with pembrolizumab was not effective in glioblastoma.[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eSeveral publications highlight the CNS penetrance and effectiveness of ICB antibodies in brain metastasis. Anti-PD1 treatment may reverse the immunosuppression within the TME and CNS tumors have been shown to respond to combined immune checkpoint blockade, resulting in elevated proportions of tumor-infiltrating lymphocytes (TILs).[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] Within the context of CAR T-cell treatment PD-1 suppression can be achieved through the co-administration of PD-1 targeting monoclonal antibodies or the PD-1 gene editing of CAR T-cells.[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIn systemic tumor models, the additional value of PD-1 blockade to increase CAR T-cell efficacy has been debated. In a murine preclinical model for systemic melanoma, concurrent PD-1 blockade notably increased the persistence and efficacy of CAR T-cell treatment.[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] However, in another study using a immunocompetent murine model for systemic melanoma, PD-1 blockade primarily mediates its anti-tumor effect through endogenous T-cells and did not increase the anti-tumor effect of CAR T-cell treatment.[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] Furthermore, it has been demonstrated, that PD-1 silencing may impair the anti-tumor function of CAR T-cells by inhibiting proliferation activity in a murine model of systemic NSCLC.[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] Song et al. demonstrated that anti-EGFRvIII CAR T-cell therapy with PD-1 checkpoint blockade in a CNS tumor model using U87 glioma cells.[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] However, it's noteworthy that these experiments were conducted in immunodeficient mice, which may overlook the influence of endogenous T-cell immunity and an intact PD-1\u0026ndash;PD-L1 signaling axis. In our study, we used autologous spleenocytes for CAR T-cell production and observe no significant difference in intratumoral densities of CAR T-cells and CD3\u003csup\u003e+\u003c/sup\u003eT-cells, nor in CAR T-cell persistence and survival following the co-administration of anti-PD-1 and CAR T-cells. Transduction with the retroviral CAR vector endows CAR T-cells with dual specificity via the CAR and the endogenous T-cell receptor (TCR). Although CAR T-cell-based therapies are recommended for the treatment of hematological malignancies, the effects of endogenous TCR signaling in CAR T-cell biology have not been well defined. Recent preclinical and clinical studies suggest that endogenous TCR signaling is not required for CAR T-cell effector function, whereas it could negatively affect proliferation and effector function[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother potential mechanism contributing to the limited efficacy of the combinatorial approach is the complex composition of the tumor microenvironment (TME) within the brain which frequently harbors fewer proliferating immune cells compared to primary tumors and other metastatic sites. Additionally, T-cells in brain metastases exhibit elevated expression levels of immune checkpoint proteins compared to those in other sites, while macrophages in the brain are more prone to expressing an immune-suppressing M2 gene signature. These factors collectively contribute to impeding the effectiveness of CAR T-cells in CNS tumors.[\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eBy utilizing repetitive in vivo TPLSM we are capable of elucidating intratumoral CAR T-cell dynamics from early stages of tumor formation until late timepoints, when large tumors have formed. After intracerebral injection of CAR T-cells, we initially observed higher intratumoral densities of EpCAM-directed CAR T-cells compared to undirected CAR T-cells. In general, CAR T-cells recognize surface antigens independently from MHC restriction. Based on the intracranial administration, early contact of EpCAM-directed CAR T-cells with EpCAM-transuced LL/2 tumor cells may lead to receptor-antigen-interaction inducing activation, proliferation and the development of a cytotoxic phenotype.[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] Interestingly, intratumoral CAR T-cell density and proliferation diminished during the observation period indicating insufficient CAR T-cell persistence within the tumor. Consequently, a decreasing amount of EpCAM-directed CAR T-cells was paralleled by tumor growth. Consistent with our data, several preclinical and clinical studies in other solid brain tumors observe decreasing CAR T-cell numbers and T-cell exhaustion even when a sufficient T-cell infiltration has been achieved.[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] Immunologically, large tumor burden requires persistent CAR T-cell function upon repeated antigen stimulation in an immunosuppressive environment to eventually achieve tumor eradication. However, chronic antigenic stimulation by the tumor results in endogenous T-cell exhaustion characterized by loss of lytic function and cytokine secretion with simultaneous expression of inhibitory receptors like PD1/PD-L1[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Consequently, we sought to elucidate the impact of concomitant anti-PD1 treatment on CAR T-cell migration and effector function. Surprisingly, we do not observe any differences in CAR T-cell migration to and persistence within the tumor after anti-PD1 treatment. In line with that, no survival differences could be observed between animals receiving ICB and the isotype control antibodies, respectively. In general, anti-PD-1 antibodies mainly function by disrupting the interaction between PD-1 on T-cells and PD-L1 on tumor cells. Paucity of PD-L1 on tumor cells is a well-defined factor associated with resistance to anti-PD-1 antibody treatment while high expression usually indicate better response rates.[\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] However, the PD-L1 expression varies among patients and between different tumor entities.[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] Furthermore, the upregulation of alternative immune checkpoints or the activation of alternative signaling pathways within tumor cells may contribute to resistance. For instance, tumor cells may exploit pathways other than the PD-1-PD-L1 axis to evade immune surveillance. Liu et al. engineered CAR T-cells by modifying PD-1, incorporating the extracellular and transmembrane domains of PD-1 with the intracellular signaling domain of CD28. This adaptation facilitated the transformation of inhibitory signals within the TME into activating signals.[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] The resultant 'switch-receptor' CAR T-cells exhibited enhanced efficacy in tumor control compared to the concurrent administration of anti-PD-1 with CAR T-cells. The conversion of multiple inhibitory signals within the TME to stimulatory signals holds significant potential for improving anti-tumor cytotoxicity. This is particularly noteworthy given the elevated expression of checkpoints, including PD-1, LAG-3, TIM-3, and TIGIT, along with their ligands, in solid brain tumors.[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] Converting the ubiquitous inhibitory signals into stimulatory signals can thereby greatly improve CAR T-cell infiltration and persistence and has to be investigated in further studies.\u003c/p\u003e \u003cp\u003eAlthough CAR T-cell therapy shows promising results in B cell malignancies, CNS affection is a common exclusion criterion in clinical trials mainly driven by fear of neurotoxicity. Additionally, most CARs targeting solid tumors use antigens shared by normal tissues, carrying the risk of on-target off-tumor toxicity. The additional use of ICB theoretically increases efficacy while also increasing the risk of toxicity. Such side effects most frequently comprise neurological symptoms, epileptic seizures, systemic immune reactions like the cytokine release syndrome (CRS), organ dysfunction and death[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Amongst others, the CAR construct in our model is constituted by an scFv capable of recognizing murine EpCAM in most epithelial tissues. As a pan-epithelial marker, EpCAM is homogenously expressed on the surface of healthy alveolar tissue[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Due to shared expression on the surface of tumor cells and healthy tissue, the risk of on-target-off-tumor reactions is significantly increased in the context auf EpCAM-directed CAR T-cells and anti-PD1 treatment[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Notably, we did not observe any clinically relevant side-effects in our fully immunocompetent mouse model. Nevertheless, it remains to be mentioned that especially due to the small sample size and the translational nature of our experimental set-up we cannot fully predict on on-target/off-tumor reactions of our combinatorial approach.\u003c/p\u003e \u003cp\u003eIn conclusion, we demonstrated that locally injected CAR T-cells adjacent to the tumor lead to intratumoral accumulation and reduced tumor growth translating into a survival benefit of \u003csup\u003eEpCAM/GFP\u003c/sup\u003eCAR T-cell treated mice. Even though additional anti-PD1 treatment was safe and well-tolerated, it does not elicit unconstrained proliferation or intratumoral persistence.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eJ.B. acknowledges research grants from the Munich Clinician Scientist Program \u0026ldquo;Else-Kr\u0026ouml;ner-Fresenius Forschungskolleg\u0026rdquo; and of the Medical Faculty of the Ludwig-Maximilians-University Munich. T.X. acknowledge scholarship support from China Scholarship Council (CSC). KJM acknowledges research grants from the Friedrich-Baur-Foundation, from the \u0026ldquo;Society for Research and Science at the Medical Faculty of the LMU\u0026rdquo; at the Ludwig-Maximilians-University Munich, and from the SFB TRR 338 (project B02). N.T. acknowledges a research grant from the \u0026quot;Support Program for Research and Teaching\u0026quot; at the Ludwig-Maximilians-University Munich. H.I-A acknowledges the SFB 914 (project Z01). P.K. acknowledges research grants from the Friedrich-Baur-Foundation, from the \u0026quot;Support Program for Research and Teaching\u0026quot; at the Ludwig-Maximilians-University Munich, from the \u0026ldquo;Society for Research and Science at the Medical Faculty of the LMU\u0026rdquo; at the Ludwig-Maximilians-University Munich, and from the \u0026ldquo;Familie Mehdorn\u0026rdquo;-Foundation. L.v.B. acknowledges support by the SFB TRR 338 (project B02) and from the advanced Munich Clinical Scientist Program\u0026rdquo; and the Helene and Bruno J\u0026ouml;ster foundation. This study was supported by the Bavarian Cancer Research Center (BZKF) (TANGO to S.K.), the Deutsche Forschungsgemeinschaft (DFG, KO5055-2-1 \u0026nbsp;and KO5055/3-1 to S.K.), the international doctoral program \u0026lsquo;i-Target: immunotargeting of cancer\u0026rsquo; (funded by the Elite Network of Bavaria; to S. K.), the Melanoma Research Alliance (grant number 409510 to S.K.), Marie Sklodowska-Curie Training Network for Optimizing Adoptive T Cell Therapy of Cancer (funded by the Horizon 2020 program of the European Union; grant 955575 to S.K.), Else Kröner-Fresenius-Stiftung (IOLIN to S.K.), German Cancer Aid (AvantCAR.de to S.K.), the Wilhelm-Sander-Stiftung (to S. K.), Ernst Jung Stiftung (to S.K.), Institutional Strategy LMUexcellent of LMU Munich (within the framework of the German Excellence Initiative; to S.K.), the Go-Bio-Initiative (to S.K.), the m4-Award of the Bavarian Ministry for Economic Affairs (to S.K.), Bundesministerium für Bildung und Forschung (to S.K.), European Research Council (Starting Grant 756017, PoC Grant 101100460 and CoG 101124203 to S.K.), by the SFB-TRR 338/1 2021\u0026ndash;452881907 (to S.K.), Fritz-Bender Foundation (to S.K.), Deutsche José Carreras Leuk\u0026auml;mie Stiftung (to S.K.), Hector Foundation (to S.K.), Bavarian Research Foundation (BAYCELLATOR to S.K.), the Bruno and Helene J\u0026ouml;ster Foundation (360\u0026deg; CAR to S.K.), the Monika-Kutzner Stiftung (to S.K.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Disclosures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJens Blobner\u003c/strong\u003e \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;- No disclosures\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLaura Dengler\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;- No disclosures\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstantin Eberle\u003c/strong\u003e \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;- No disclosures\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJulika Herold\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e- No disclosures\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTao Xu\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;- No disclosures\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlexander Beck\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;- No disclosures\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnton M\u0026uuml;hlbauer\u003c/strong\u003e \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;- No disclosures\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKatharina Mueller\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;- No disclosures\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhilipp Karschnia\u003c/strong\u003e \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;- No disclosures\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNico Teske\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;- No disclosures\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDominic van den Heuvel\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;- No disclosures\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFerdinand Schallerer\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;- No disclosures\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHellen Ishikawa-Ankerhold\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;- No disclosures\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMarion Subklewe\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;- No disclosures\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNiklas Thon\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;- No disclosures\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ\u0026ouml;rg-Christian Tonn\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;- Research grants from Novocure and Munich Surgical Imaging; and Royalties from Springer Publisher Intl.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMarion Subklewe\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;- No disclosures\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSebastian Kobold\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;-\u0026nbsp;S. K. has received honoraria from TCR2 Inc., Miltenyi, Galapagos, Novartis, BMS and GSK. S. K. is an inventor of several patents in the field of immuno-oncology. S. K. received license fees from TCR2 Inc and Carina Biotech. S.K. received research support from TCR2 Inc., Tabby Therapeutics, Catalym GmBH, Plectonic GmBH and Arcus Bioscience for work unrelated to the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatrick N. Harter\u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;- No disclosures\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVeit R Buchholz\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;- No disclosures\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLouisa von Baumgarten\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; - No disclosures\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.B., L.v.B. conceptualization, planning and drafting of the manuscript J.B., L.D. performed the main experiments. 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OncoImmunology 9:1806009. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/2162402X.2020.1806009\u003c/span\u003e\u003cspan address=\"10.1080/2162402X.2020.1806009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"CAR T cells, brain metastasis, lung cancer, PD1-blockade","lastPublishedDoi":"10.21203/rs.3.rs-4456398/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4456398/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eLung cancer brain metastasis have a devastating prognosis, necessitating innovative treatment strategies. While chimeric antigen receptor (CAR) T-cells show promise in hematologic malignancies, their efficacy in solid tumors, including brain metastasis, is limited by the immunosuppressive tumor environment. The PD-L1/PD-1 pathway inhibits CAR T-cell activity in the tumor microenvironment, presenting a potential target to enhance therapeutic efficacy. This study aims to evaluate the impact of anti-PD1 antibodies on CAR T-cells in treating lung cancer brain metastasis.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe utilized a murine immunocompetent, syngeneic orthotopic cerebral metastasis model for repetitive intracerebral two-photon laser scanning microscopy (TPLSM), enabling in vivo characterization of red fluorescent tumor cells and CAR T-cells at a single-cell level over time. Red fluorescent EpCAM-transduced Lewis Lung carcinoma cells (\u003csup\u003eEpCAM/tdt\u003c/sup\u003eLL/2 cells) were implanted intracranially. Following the formation of brain metastasis, EpCAM-directed CAR T-cells were injected into adjacent brain tissue, and animals received either anti-PD-1 or an isotype control.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCompared to controls receiving T-cells lacking a CAR, mice receiving EpCAM-directed CAR T-cells showed higher intratumoral CAR T-cell densities in the beginning after intraparenchymal injection. This finding was accompanied with reduced tumor growth and translated into a survival benefit. Additional anti-PD1 treatment, however, did not affect intratumoral CAR T-cell persistence nor tumor growth and thereby did not provide an additional therapeutic effect.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eCAR T-cell therapy for brain malignancies appears promising. However, additional anti-PD1 treatment did not enhance intratumoral CAR T-cell persistence or effector function, highlighting the need for novel strategies to improve CAR T-cell therapy in solid tumors.\u003c/p\u003e","manuscriptTitle":"PD-1 blockade does not improve efficacy of EpCAM-directed CAR T-cells in lung cancer brain metastasis.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-03 15:21:32","doi":"10.21203/rs.3.rs-4456398/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-15T22:14:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-09T18:10:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-29T02:49:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"158735536490059632338020832401918204648","date":"2024-05-28T19:34:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-27T03:26:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"233133788597013283036337021867458842392","date":"2024-05-26T19:59:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"86178902191520551150509131527249869859","date":"2024-05-26T18:00:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-26T17:33:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-22T05:01:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-22T05:01:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Immunology, Immunotherapy","date":"2024-05-21T17:26:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"55c59430-a23a-4289-bf4f-ed16c6537ff4","owner":[],"postedDate":"June 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-07T16:05:47+00:00","versionOfRecord":{"articleIdentity":"rs-4456398","link":"https://doi.org/10.1007/s00262-024-03837-9","journal":{"identity":"cancer-immunology-immunotherapy","isVorOnly":false,"title":"Cancer Immunology, Immunotherapy"},"publishedOn":"2024-10-03 15:58:22","publishedOnDateReadable":"October 3rd, 2024"},"versionCreatedAt":"2024-06-03 15:21:32","video":"","vorDoi":"10.1007/s00262-024-03837-9","vorDoiUrl":"https://doi.org/10.1007/s00262-024-03837-9","workflowStages":[]},"version":"v1","identity":"rs-4456398","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4456398","identity":"rs-4456398","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}