Targeted degradation of CD24 by a transferrin receptor-engaging bispecific degrader enhances antitumor immunity.

Glioblastoma (GBM) is the most common malignant diffuse glioma of the brain. Although immunotherapy with immune checkpoint inhibitors (ICIs), such as programmed cell death protein (PD)‐1/PD ligand‐1 inhibitors, has revolutionized the treatment of several cancers, the clinical benefit in GBM patients has been limited. Lymphocyte‐activation gene 3 (LAG‐3) binding to human leukocyte antigen‐II (HLA‐II) plays an essential role in triggering CD4+ T cell exhaustion and could interfere with the efficiency of anti‐PD‐1 treatment; however, the value of LAG‐3–HLA‐II interactions in ICI immunotherapy for GBM patients has not yet been analyzed. Therefore, we aimed to investigate the expression and regulation of HLA‐II in human GBM samples and the correlation with LAG‐3+CD4+ T cell infiltration. Human leukocyte antigen‐II was highly expressed in GBM and correlated with increased LAG‐3+CD4+ T cell infiltration in the stroma. Additionally, HLA‐IIHighLAG‐3High was associated with worse patient survival. Increased interleukin‐10 (IL‐10) expression was observed in GBM, which was correlated with high levels of HLA‐II and LAG‐3+ T cell infiltration in stroma. HLA‐IIHighIL‐10High GBM associated with LAG‐3+ T cells infiltration synergistically showed shorter overall survival in patients. Combined anti‐LAG‐3 and anti‐IL‐10 treatment inhibited tumor growth in a mouse brain GL261 tumor model. In vitro, CD68+ macrophages upregulated HLA‐II expression in GBM cells through tumor necrosis factor‐α (TNF‐α). Blocking TNF‐α‐dependent inflammation inhibited tumor growth in a mouse GBM model. In summary, T cell–tumor cell interactions, such as LAG‐3–HLA‐II, could confer an immunosuppressive environment in human GBM, leading to poor prognosis in patients. Therefore, targeting the LAG‐3–HLA‐II interaction could be beneficial in ICI immunotherapy to improve the clinical outcome of GBM patients.

Endocytosis is an essential membrane transport mechanism that is indispensable for the maintenance of life. It is responsible for the selective internalization and subsequent degradation or recycling of specific extracellular proteins and nutrients, thereby facilitating cellular nutrient supply, modulation of receptor signaling, and clearance of foreign substances. However, methods for the quantitative analysis of lysosomal degradation of extracellular proteins via endocytosis remain limited. This protocol describes a method for purifying the protein-of-interest (POI)–red fluorescent protein (RFP)–green fluorescent protein (GFP) fusion protein, which is modified with specific mammalian cell glycans or other modifications, from the conditioned medium of mammalian cell cultures. Subsequently, the protocol details a quantitative approach for evaluating its internalization and lysosomal degradation within cells using the RFP–GFP tandem fluorescent reporter. Following the addition of POI-RFP-GFP to the medium, cells can be subjected to cell biological assays, such as flow cytometry, as well as biochemical analyses, such as immunoblotting. This protocol is broadly applicable to studies of the internalization of extracellular proteins.Key features• Purification of secreted GFP-RFP-fused POI from mammalian cell culture supernatant.• Quantification of POI-RFP-GFP internalization through measurement of GFP and RFP signals using flow cytometry.• Confirmation of lysosomal degradation of POI-RFP-GFP by immunoblotting.

Glioblastoma (GBM) is a highly malignant brain cancer with a poor prognosis despite standard treatments. This investigation aimed to explore the feasibility of PTPN6 to combat GBM with immunotherapy. Our study employed a comprehensive analysis of publicly available datasets and functional experiments to assess PTPN6 gene expression, prognostic value, and related immune characteristics in glioma. We evaluated the influence of PTPN6 expression on CD8+ T cell exhaustion, immune suppression, and tumor growth in human GBM samples and mouse models. Our findings demonstrated that PTPN6 overexpression played an oncogenic role in GBM and was associated with advanced tumor grades and unfavorable clinical outcomes. In human GBM samples, PTPN6 upregulation showed a strong association with immunosuppressive formation and CD8+ T cell dysfunction, whereas, in mice, it hindered CD8+ T cell infiltration. Moreover, PTPN6 facilitated cell cycle progression, inhibited apoptosis, and promoted glioma cell proliferation, tumor growth, and colony formation in mice. The outcomes of our study indicate that PTPN6 is a promising immunotherapeutic target for the treatment of GBM. Inhibition of PTPN6 could enhance CD8+ T cell infiltration and improve antitumor immune response, thus leading to better clinical outcomes for GBM patients.

Background: Glioblastoma (GBM), the most common and lethal primary brain tumor, has a median survival of a mere 15 months and leads to approximately 12,000 deaths in the US annually. Targeted and combinatorial-based clinical trial therapies have shown poor efficacy in GBM treatment, partly due to the restrictive nature of the blood-brain barrier, an immunosuppressive tumor microenvironment, GBM’s heterogeneity and adaptability, and GBM’s ability to metastasize and invade critical regions of the brain. However, promising recent literature has indicated that neoadjuvant anti-PD-1 checkpoint-inhibition immunotherapy - i.e., starting it right before surgery for recurrence - improves survival outcomes in human GBM patients. Results: Here, we demonstrate a proposed mechanism of action wherein localized intratumoral danger-associated molecular pattern (DAMP, a known immunogenic driver) injection of calreticulin - used to mimic natural DAMP release from necrotic cells during surgery - combined with neoadjuvant anti-PD-1 immunotherapy leads to better survival outcomes in both orthotopic mouse CT2A and CT2A-Luc GBM models. This survival benefit is also seen in a more aggressive (larger tumor inoculation size) orthotopic CT2A-Luc GBM model. Flow cytometry indicates increased microglia cell counts and activation marker expression, and increased myeloid activation marker expression in mice brains treated with our combination immunotherapy in a CT2A GBM model. Additionally, in vivo treatment with our combination immunotherapy led to increases in the local T and NK cell numbers, the CD8:CD4 ratio, and the proliferation of CD4 T cells in mice brains of a CT2A GBM model. In vitro results suggest that co-culture with CT2A cells increased PD-1 expression in macrophages and microglia and that our combination treatment of calreticulin and anti-PD-1 immunotherapy reduces the viability of mouse GBM cells when mixed with macrophages. Significance: This project paves the path for a novel immunotherapeutic approach to tackle GBM and other cancers. Future studies could incorporate relevant DAMP’s into nanoparticles for sustained release after intratumoral injection and possibly viral delivery of DAMP’s that are constitutively secreted, thereby prolonging an anticipated immune response. Citation Format: Suchet Taori, Breanna Noffsinger, Charlotte A. Miller, Aizhen Xiao, Laryssa Manigat, Qing Zhong, Tajie Harris, Benjamin Purow. Staged anti-PD-1 therapy with intratumoral recombinant calreticulin improves anti-tumor immunity and survival in glioblastoma mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4205.

Glioblastoma (GBM) is the most aggressive and common type of brain tumor in adults, with patient survival times of approximately one year following diagnosis. The GBM microenvironment is composed of numerous non-neoplastic cells, including vascular endothelia, various infiltrating and resident immune cells, and non-neoplastic glial cells. The most abundant non-neoplastic cell population in the GBM microenvironment is tumor-associated macrophages (TAMs). TAMs comprise mixed populations of myeloid cells, including infiltrating macrophages from the blood circulation and resident brain microglia. TAMs are recruited to the GBM microenvironment, where they are hypothesized to perform immunosuppressive functions and release growth factors and cytokines in response to factors produced by neoplastic cells. In an effort to delineate the temporal and spatial dynamics of TAM composition during malignant gliomagenesis, we employed genetically-engineered mouse models of PDGF-driven GBM in combination with double-transgenic reporter mice that express GFP (green fluorescent protein) or RFP (red fluorescent protein) under the control of CX3CR1 or CCR2 promoters, respectively. Using this approach, we demonstrated that CX3CR1LoCCR2Hi monocytes are recruited to the GBM, where they transition in situ to CX3CR1HiCCR2Lo macrophages and CX3CR1HiCCR2- microglia-like cells. We found that infiltrating macrophages constitute ~80% of the total TAM population, with resident microglia accounting for the remaining ~20% of TAMs. Bone marrow-derived infiltrating cells (macrophages) preferentially localize to perivascular areas. In contrast, resident microglia are mainly localized to peri-tumoral regions. Additionally, RNA-sequencing analyses revealed differential gene expression patterns unique to infiltrating and resident cells, suggesting unique functions for each TAM population. Collectively, these results demonstrate the unique spatial and temporal differential composition, as well as distinct anatomical localizations for infiltrating and resident monocytes in GBM. Our study provides a strong rationale for targeting infiltrating cells, the major component of TAMs, in future stroma-directed therapeutic approaches against this deadly neoplasm.

Background: Glioblastoma (GB) is a particularly malignant brain tumour which carries a poor prognosis and presents limited treatment options. MRI is standard practice for differential diagnosis at initial presentation of GB and can assist in both treatment planning and response assessment. MRI radiomics allows for discerning GB features of clinical importance that are not evident by visual analysis, augmenting the morphological and functional tumour characterisation beyond traditional imaging techniques. Given that radiotherapy is part of the standard of care for GB patients, establishing a platform for phenotyping radiation treatment responses using non-invasive methods is of high relevance. Methods: In this study, we modelled the responses to ionising radiation across four orthotopic mouse models of GB using diffusion and perfusion radiomics. We have identified the optimal set of radiomic features that reflect tumour cellularity, microvascularity, and blood flow changes brought about by radiation treatment in these murine orthotopic models of GB, and directly compared them with endpoint histopathological analysis. Results: We showed that the selected radiomic features can quantify textural information and pixel interrelationships of tumour response to radiation therapy, revealing subtle image patterns that may reflect intra-tumoural spatial heterogeneity. When compared to GB patients, similarities in selected radiomic features were noted between orthotopic murine tumours and non-enhancing central tumour areas in patients, along with several discrepancies in tumour cellularity and vascularization, denoted by distinct grey level intensities and nonuniformity metrics. Conclusion: As the field evolves, radiomic profiling of GB may enhance the evaluation of targeted therapeutic strategies, accelerate the development of new therapies, and act as a potential virtual biopsy tool.

Glioblastoma (GBM) is a common and aggressive brain cancer that accounts for 60% of adult brain tumors. Anti-angiogenesis therapy is an attractive option due to the high vasculature density of GBM. However, the best-known anti-angiogenic therapeutics, bevacizumab, and aflibercept, have failed to show significant benefits in GBM patients. One of the reasons is the limited brain penetration of antibody-based therapies due to existence of the blood–brain barrier (BBB), which is further strengthened by the blood vessel normalization effects induced by anti-angiogenic therapies. To investigate if increased drug concentration in the brain by transferrin receptor (TfR)-mediated delivery across the BBB can enhance efficacy of anti-angiogenic antibody therapies, we first identified an antibody that binds to the apical domain of the mouse TfR and does not compete with the natural ligand transferrin (Tf) binding to TfR. Then, we engineered two bispecific antibodies fusing a vascular endothelial growth factor (VEGF)-Trap with the TfR-targeting antibody. Characterization of the two bispecific formats using multiple in vitro assays, which include endocytosis, cell surface and whole-cell TfR levels, human umbilical vein endothelial cell growth inhibition, and binding affinity, demonstrated that the VEGF-Trap fused with a monovalent αTfR (VEGF-Trap/moAb4) has desirable endocytosis without the induction of TfR degradation. Peripherally administered VEGF-Trap/moAb4 improved the brain concentration of VEGF-Trap by more than 10-fold in mice. The distribution of VEGF-Trap/moAb4 was validated to be in the brain parenchyma, indicating the molecule was not trapped inside the vasculature. Moreover, improved VEGF-Trap brain distribution significantly inhibited the angiogenesis of U-87 MG GBM tumors in a mouse model.

Lysosome-targeting chimeras enable targeted protein degradation.

Radioresistance remains a major clinical challenge in glioblastoma (GB) therapy. However, the mechanism for the development of radioresistance in GB is unclear. Mounting evidence suggests that Galectin-1 (Gal1: a carbohydrate-binding protein galectin-1) contributes to the radioresistance of GB tumors. Recently, several Gal1-targeting compounds have emerged. OTX008 is a calixarene derivative designed to bind the Gal1 amphipathic β-sheet conformation. Our study aimed to reduce galectin-1 expression using OTX008 in a human GB pre-clinical model. For these studies an in vivo GL261 murine models of GB were utilized to assess efficacy of treatment with radiation plus OTX008. Our results demonstrated that inhibition of Gal-1 with OTX008 plus radiation showed significantly decrease in GB growth rate and improved survival in in vivo. OTX008 treatment plus radiation was associated with downregulation of Gal1 and Ki67 in treated tumors, as well as decreased microvessel density and VEGFR2 expression. Finally, combination studies showed OTX008 synergy with radiation therapies, principally when OTX008 was administered first. This study provides insights of Gal-1inhibitory effects of OTX008 and its enhancement of the efficacy of radiotherapy in mouse models of GB suggest that further studies are warranted.

Transdifferentiation (TD) is an exciting new advancement in somatic cell reprogramming that eliminates the pluripotent intermediate stage to create cells that are ideal for personalized cell transplant therapy. Induced neural stem cells (iNSCs) are the newest cell type created by TD. Here we begin to define the efficacy of iNSC therapy for central nervous system disorders. We developed the first iNSC-based drug deliver vehicles, and show that engineered iNSCs are tumor-homing drug delivery vehicles with significant anti-cancer effects in models of glioblastoma (GBM). After genetically engineering iNSCs with tumoricidal or diagnostic transgenes, we observed that the modified iNSCs proliferated and differentiated into astrocytes and neurons with the same efficiency as unmodified cells. Non-invasive serial imaging revealed that engineered iNSCs implanted into the parenchyma of mice survived more than 1 month. In vitro time-lapse motion analysis showed iNSCs exhibit tumor-homing properties similar to brain-derived NSCs, while iNSCs implanted into the frontal lobe of mice migrated through the brain homing to invasive GBM cells. iNSCs engineered with the anti-cancer molecule TRAIL (iNSC-sTR) stably released the tumoricidal agent and killed co-cultured human GBM cells. We evaluated iNSC-sTR therapy in orthotopic mouse models of solid and patient-derived diffuse GBM. We found that iNSC-sTR therapy reduced GBM volumes 20- to 230-fold and doubled the survival of tumor-bearing mice compared to control mice. These data provide the first evidence that tumoricidal iNSCs can be used to efficaciously treat brain cancer, and provide a foundation for the continued development of iNSC-based therapies.

Pediatric Glioblastoma (GBM) remains one of the most difficult childhood tumors to treat, and most children with this diagnosis will not survive longer than two years. ATRX is a histone chaperone protein that is mutated primarily in pediatric patients with GBM and younger adults with secondary GBM. No previous animal model has demonstrated the effect of ATRX loss on GBM formation. We cloned an ATRX knockdown sequence into a Sleeping Beauty (SB) transposase-responsive plasmid (shATRX) for insertion into host genomic DNA. Glioblastomas were induced in neonatal mice by injecting plasmids encoding SB transposase/ luciferase, shp53 and NRAS, with or without shATRX, into the ventricle of neonatal mice. Tumors in both groups (with or without shATRX) showed histological hallmarks of human glioblastoma. The loss of ATRX was specifically localized only within tumors generated with the shATRX plasmid and not in the adjacent cortex. Notably, loss of ATRX reduced median survival of mice by 43% (p = 0.012). ATRX-deficient tumors displayed evidence of telomeric lengthening using telomeric FISH assay for alternative lengthening of telomeres (ALT). ATRX-deficient tumors were significantly more likely to develop microsatellite instability (p = 0.014), a hallmark of impaired DNA-damage repair. Analysis of three human GBM sequencing datasets confirmed increased number of somatic nucleotide mutations in ATRX-deficient tumors. Treatment of primary cell cultures generated from mouse GBMs showed that ATRX-deficient tumor cells are significantly more sensitive to certain DNA damaging agents, with greater evidence of double-stranded DNA breakage, by gH2A.X. In addition, mice with ATRX-deficient GBM treated with whole brain irradiation showed reduced tumor growth by luminescence, with some long-term survivors. In summary, this mouse model prospectively validates ATRX as a tumor suppressor in human GBM for the first time in an animal model. In addition, loss of ATRX leads to increased genetic instability and response to DNA-damaging therapy. Based on these results, we have generated the hypothesis that ATRX loss leads to a genetically unstable tumor; which is more aggressive when untreated, but more responsive to DNA-damaging therapy, ultimately resulting in equivalent or improved overall survival. Supported by St. Baldrick's Fellowship and Alex's Lemonade Stand /Northwest Mutual Young Investigator Award to CK and NIH/NINDS grants to MGC and PRL. Citation Format: Carl Koschmann, Alexandra Calinescu, Daniel Thomas, Felipe J. Nunez, Marta Dzaman, Johnny Krasinkiewicz, Rosie Lemons, Neha Kamran, Flor Mendez, Soyeon Roh, David Ferguson, Pedro R. Lowenstein, Maria G. Castro. ATRX validated as tumor suppressor in a novel mouse model of pediatric and young adult GBM. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3009. doi:10.1158/1538-7445.AM2015-3009

911 Anti-VEGF therapy improves EGFR-vIII-CAR-T cell delivery and efficacy in syngeneic glioblastoma models in mice

Evaluating an immunotherapeutic approach to photodynamic therapy for glioblastoma.

Most patients with recurrent glioblastoma (GBM) are treated with bevacizumab, a humanized monoclonal antibody (mAb) that binds VEGF-A and inhibits its binding to VEGFR. Approximately 30% of GBM patients are non-responsive to bevacizumab and the underlying mechanism for the lack of response is not known. It has been assumed that bevacizumab solely targets circulating VEGF-A in blood. We hypothesized that bevacizumab and human IgGs in general gain access to the perivascular niche that contains cancer stem cells (CSCs) in GBM. We found that bevacizumab gains access to the perivascular tumor area through leaky blood vessels and was internalized by tumor cells in an orthotopic xenograft mouse model of GBM. In vitro, CSCs (CD133+) from GBM rapidly internalized either bevacizumab or human IgG into membrane protrusions that contained actin and internalization was significantly inhibited by a macropinocytosis inhibitor (EIPA), suggesting CSCs internalize bevacizumab or human IgG via macropinocytosis. Furthermore, bevacizumab or human IgG was largely detected in the Rab4+ “fast” recycling compartment at 5 min, and both were largely detected in the LAMP1+ compartment (late endosome/lysosome) at 3 hr in the CSCs. CSCs (CD133+) from GBM do not express the neonatal Fc receptor, the canonical pathway for recycling of IgG. Administration of bevacizumab to an orthotopic xenograft mouse model of established GBM showed that bevacizumab was partially co-localized with Rab4+ or with LAMP1+ in perivascular tumor cells, consistent with our in vitro findings. Taken together, our data show that in GBM, humanized IgG, including bevacizumab, gains access to the perivascular tumor space and is then macropinocytosed by CSCs and trafficked to a recycling compartment or to the late endosome/lysosome. These data suggest that alterations in endocytosis or recycling in the CSCs could impact the fate of therapeutic IgGs like bevacizumab and ultimately influence a patients’ response to GBM therapy. Citation Format: Gaelle Muller-Greven, Cathleen Carlin, Steven Toms, Manmeet Ahluwalia, Markus Bredel, Justin Lathia, Jeremy Rich, Petra Hamerlik, Candece L. Gladson. The tissue and cellular destination of therapeutic IgGs in glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3276.

OBJECTIVE A new imaging technology that indiscriminately detects intracranial glioblastoma (GBM) can help neurosurgeons remove tumor mass completely. Transferrin receptors (TfR 1) have been widely investigated as a diagnostic and therapeutic target in GBM. A TfR 1-targeted peptide, CRTIGPSVC (CRT) can accumulate at high levels in GBM tissues. In our study, taking the advantage of CRT, we synthesized two molecular imaging probes for imaging GBM precisely. One is a PET/CT probe 18F-NOTA-CRT, and the other is a near-infrared fluorescent (NIFR) probe Cy5-CRT. METHODS We initially confirmed the overexpression of TfR 1 in most of GBM and the tumor-specific homing ability of 18F-NOTA-CRT and Cy5-CRT in orthotopic U87 GBM (TfR 1 overexpression) mouse models. We then examined the feasibility of Cy5-CRT for specially identifying the GBM tissue margin in the intracranial U87 xenografts in vivo and ex vivo. Next, we compared Cy5-CRT with the clinically used fluorescein sodium in identifying tumor margins. Finally, we used Cy5-CRT to carry out a fluorescence-guided operation on a orthotopic U87 mouse model. RESULTS Both 18F-NOTA-CRT and Cy5-CRT probes specifically accumulated in U87 GBM xenografts with TfR 1 overexpression, but not in U373 GBM xenografts with very low TfR 1 expression. Cy5-CRT detected the intracranial tumor burden with exceptional contrast, enabling fluorescence-guided GBM resection under NIFR live imaging conditions. Importantly, Cy5-CRT recognized the GBM tissue margin more clearly than fluorescein sodium. CONCLUSIONS Our probes were capable of thoroughly detecting GBM tissue in vivo imaging. For translational applications, we may screen patients before surgery by PET/CT imaging with 18F-NOTA-CRT to identify gliomas with TfR 1 overexpression. As for fluorescence-guided surgery, the TfR 1-targeted optical probe Cy5-CRT specifically differentiates tumor tissues from normal brain with high sensitivity, indicating its potential application for the precise surgical removal of GBM. Keywords: Transferrin receptor 1; PET/CT; near-infrared fluorescence imaging; glioblastoma, fluorescence-guided surgery