Treatment Of Tumor-bearing Mice Research Articles

Daidzein-directed methionine γ-lyase in enzyme prodrug therapy against breast cancer

Thiosulfinates in situ formed by “pharmacological pair” C115H methionine γ-lyase/S-(allyl/alkyl)-l-cysteine sulfoxides possess cytotoxic activity against various malignant cell lines. To investigate in vivo antitumor activity of thiosulfinates generated directly at the surface of tumor cells, a chemical conjugate between Clostridium novyi C115H methionine γ-lyase (C115H MGL) and isoflavone daidzein was prepared. The binding of conjugate (C115H-Dz) to various breast cancer cell lines was demonstrated, as well as its cytotoxicity in the presence of S-(allyl/alkyl)-l-cysteine sulfoxides. The most promising among thiosulfinates was dipropyl thiosulfinate (IC50 < 0.53 μM). The pharmacokinetic parameters of C115H MGL and C115H-Dz were obtained. Plasma half-lives of the enzyme and conjugated enzyme were 4.4 and 7.2 h, respectively. In vivo antitumor effect of pharmacological pairs on SKBR-3 xenografts was demonstrated. Treatment of tumor-bearing mice with a pair of C115H-Dz/propiin inhibited tumor growth by 85%.

Abstract LB024: Cancer stem cells are enriched in human recurrent glioblastoma

Abstract A number of studies have described an essential role of cancer stem cells (CSC) in glioblastoma (GBM) progression and recurrence based on isolation by candidate markers. However, a systematic evaluation of the proposed CSC in paired human primary and recurrent glioblastoma has been lacking and whether they represent a unified cell type with conserved features over tumor evolution is unresolved. In this study, we used human patient derived xenografts (PDX) to investigate the status of these cells in patients following therapeutics and recurrence. Firstly, to ensure the fidelity of tumor heterogeneity in serially passaged orthotopic GBM PDX, we collected samples at different passages and performed whole tumor single cell RNA sequencing analysis. The results illustrate the preservation of all unsupervised transcriptional cell clusters through multiple passages. Furthermore, treatment of tumor bearing mice with temozolomide, the standard of care chemotherapeutic drug for glioblastoma patients, increased apoptosis and decreased tumor cell burden but spared CSC. We then turned to paired human primary and recurrent GBM samples to inquire cancer stem cell marker expression and cell distribution. In all three pairs of PDX studied, cancer stem cell marker F3 serves as a reliable means to isolate sphere formation cells and is augmented in the recurrent samples. Inspection of whole tumors with single cell RNA sequencing nails down the enrichment of CSC in recurrent tumors, thus emphasizing the importance to eradicate these cells in future therapeutics. Citation Format: Xuanhua Peter Xie, Mungunsarnai Ganbold, Alicia Pedraza, Luis Parada. Cancer stem cells are enriched in human recurrent glioblastoma [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 LB024.

Abstract 3309: Self-assembling and disassembling (SADA) domain is critical to the binding, and anti-tumor efficacy of GD2-SADA

Abstract Introduction: GD2-SADA is a pre-targeted radioimmunotherapy drug-candidate designed to target radioactive lutetium-177 (in the form of 177Lu-DOTA) to GD2-expressing tumor cells (GD2-SADA:177Lu-DOTA Drug Complex). GD2-SADA is comprised of an anti-GD2 single-chain variable fragment (scFv), an anti-DOTA scFv that binds specifically to DOTA metal chelates, such as 177Lu-DOTA, and a SADA domain based on the human P53 tetramerization sequence, which can self-assemble from a monomeric 60-kDa polypeptide into 240-kDa tetrameric protein. We have previously demonstrated (1) that GD2-SADA has a unique clearance profile that allowed for the delivery of 177Lu-DOTA to tumors with minimal exposure to normal tissues like the bone marrow or kidneys and shrank established neuroblastoma in preclinical mouse models. We now provide additional mechanistic data demonstrating the importance of the SADA domain in providing this potent anti-tumor function. Methods: To study the role of the SADA domain, we designed a modified version of GD2-SADA that could not self-assemble into a tetrameric state by removing the entire SADA domain. The resulting P53(-/-)GD2-SADA protein retained binding to GD2 and Lu-DOTA but remained in a 60 kDa monomeric state in assays. P53(-/-)GD2-SADA and GD2-SADA were compared using SPR to measure antigen binding affinities, flow cytometry to evaluate cell binding, and in preclinical mouse models to evaluate tumor uptake and anti-tumor efficacy. Results: GD2-SADA demonstrated stronger binding affinity to GD2 antigen compared to P53(-/-)GD2-SADA, but comparable binding affinity to Lu-DOTA. In SPECT/CT imaging studies using tumor bearing mice, GD2-SADA demonstrated substantially higher uptake and persistence in GD2-expressing tumors compared with P53(-/-)GD2-SADA. Finally, treatment of tumor bearing mice with 3 cycles of GD2-SADA or P53(-/-)GD2-SADA and 177Lu-DOTA (once per week each for 3 weeks) resulted in potent anti-tumor responses from GD2-SADA, with only modest anti-tumor efficacy from P53(-/-)GD2-SADA at two different dose levels corresponding to equal molar doses (1 GD2-SADA to 1 P53(-/-)GD2-SADA) or equal mass doses (1 GD2-SADA to 4 P53(-/-)GD2-SADA i.e. same number of binding sites). Conclusions: The tetramerizing function of the SADA domain is critically important to the binding activity and anti-tumor efficacy of GD2 SADA. These data confirm that the SADA domain increases tumor antigen binding, uptake and persistence in tumor tissue, and markedly improves anti-tumor responses in preclinical models.

Abstract 5542: Characterization of a long-acting IL-13 super-antagonist engineered to target tumor associated macrophages and myeloid cells

Abstract Introduction: Regulation of Th1/Th2 balance in the tumor microenvironment (TME) plays an important role in both tumor progression and response to immunotherapy. The IL-4/IL-13 pathway induces an anti-inflammatory (Th2) response by stimulating M2 skewing of tumor associated macrophage (TAM) and myeloid derived suppressor cells (MDSCs), thereby supporting tumor growth by suppressing Th1 response. Therefore, inhibition of the IL-4/IL-13 pathway has the potential to inhibit tumor growth by shifting the TME towards a pro-inflammatory response. To achieve this, we engineered a long-acting IL-13 super-antagonist (i.e., Fc-MDNA413) to target the IL-13Rα1 component of type II IL-4 receptor (IL-4Rα/IL-13α1) expressed on TAMs and MDSCs to inhibit their differentiation and expansion for immuno-oncological applications. Experimental Procedure: Studies conducted include binding analyses by Biacore/SPR, signaling analyses in IL-4/IL-13 cell reporters, in vitro cell proliferation, in vitro M1/M2 macrophage polarization and in vivo efficacy in syngeneic tumor models. Summary of Data: IL-13Rα1 and IL-4Rα constitute the type II IL-4R that drives a Th2 response upon activation by IL-4 and IL-13. Binding studies showed that in comparison to wild-type Fc-IL13, Fc-MDNA413 has higher affinity for IL-13Rα1 but lower affinity for the decoy IL-13Rα2 receptor, indicating a more favorable receptor selectivity. In a cell-based reporter assay, Fc-MDNA413 inhibits both IL-4 and IL-13 induced p-STAT6 signaling, a pathway that drives towards a Th2 response. The antagonism of Fc-MDNA413 was also observed in an IL-13 dependent TF-1 proliferation assay where dose-dependent inhibition of cell proliferation was demonstrated. In a macrophage polarization assay with human primary myeloid cells, Fc-MDNA413 inhibited both IL-4 and IL-13 induced M2 polarization in a dose-dependent manner, consistent with an inhibitory effect on IL-4/IL-13 signaling. Treatment of tumor bearing mice with Fc-MDNA413 inhibited tumor growth in multiple syngeneic tumor models (e.g., EMT6 breast cancer and KLN205 lung cancer) known to have higher percentage of TAMs and MDSCs in TME, hence demonstrating the therapeutic potential of this long-acting IL-4/IL-13 super-antagonist. Conclusion: Fc-MDNA413 is a long-acting IL-4/IL-13 super-antagonist that inhibits IL-4 and IL-13 induced signaling and M2 skewing of macrophages, resulting in tumor growth inhibition in vivo. Ongoing studies include testing in additional syngeneic tumor models, deciphering in vivo mechanism of action and investigating potential synergy with other therapies. Next generation multifunctional superkines that incorporate IL-4/IL-13 antagonism are under development to target immunologically challenging tumors. Citation Format: Aanchal Sharma, Manjunatha Ankathatti Munegowda, Fahar Merchant, Minh D. To. Characterization of a long-acting IL-13 super-antagonist engineered to target tumor associated macrophages and myeloid cells [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 5542.

Abstract 2166: Ibuprofen induces ferroptosis to potentiate antitumor immunity

Abstract Ferroptosis is an iron-dependent regulated cell death pathway that has recently emerged as a promising target for cancer therapy. PTGS2 has been identified as a marker of ferroptosis due to its upregulation in cells after treatment with ferroptosis inducers. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the protein product of PTGS2 and have been shown to enhance antitumor immunity. We hypothesized that NSAIDs induce ferroptosis and that this mechanism is essential for their effect on antitumor immunity. To test this, we first determined that ibuprofen and other NSAIDs induce cell death in YUMMER-1.7 melanoma and MC38 colorectal cancer cells in vitro. Cell death occurred progressively over 24 hours and could be blocked by multiple ferroptosis inhibitors. Treatment with NSAIDs also increased intracellular lipid peroxides and decreased levels of reduced glutathione and ferrous iron. This indicated that ibuprofen and other NSAIDs induce ferroptosis in vitro, so we next evaluated whether ibuprofen also induces ferroptosis in vivo. Analysis of ibuprofen-treated YUMMER-1.7 or MC38 tumors showed increased levels of lipid peroxides and lipid peroxide intermediates, 4-hydroxynonenal and malondialdehyde, compared to untreated tumors. Treatment of tumor-bearing mice with ibuprofen alone resulted in approximately 60% tumor clearance and survival, whereas a combination of multiple ferroptosis inhibitors with ibuprofen completely abrogated this effect. Since it has been demonstrated by that CD8+ T cells can release IFN-γ to induce ferroptosis in tumor cells, we next tested the dependence of the antitumor effect of ibuprofen on CD4+ T cells, CD8+ T cells, and IFN-γ using depleting antibodies. Depletion of any of these was sufficient to prevent the antitumor effect of ibuprofen. Given the need to identify pathways that enhance responses to existing immunotherapies, we determined whether treatment with ibuprofen synergizes with PD-1 checkpoint blockade. The combination of α-PD-1 with ibuprofen resulted in a 100% tumor clearance and survival rate, whereas α-PD-1 or ibuprofen alone resulted in 40% and 60% tumor clearance and survival rates, respectively. Furthermore, treatment with ibuprofen also sensitized α-PD-1-resistant tumors to PD-1 in two distinct models of α-PD-1 resistance. Overall, these findings establish ibuprofen and other NSAIDs as inducers of ferroptosis and underscore the potential for ferroptosis inducers to be used in combination with existing immunotherapy regimens. Citation Format: Ronan Talty, Michelle Ferreira, Irina Krykbaeva, Marcus Bosenberg. Ibuprofen induces ferroptosis to potentiate antitumor immunity [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 2166.

Abstract 2883: Dectin-2 agonist antibodies reprogram tumor-associated macrophages to drive anti-tumor immunity

Abstract Tumor-associated macrophages (TAMs), an abundant immune cell population in most cancers, support tumor progression through their immunosuppressive effects. We discovered that TAMs express the pattern recognition receptor Dectin-2 (Clec4n/CLEC6A), an activating C-type lectin receptor (CLR) that binds to high-mannose glycans on fungi and other microbes and stimulates immune responses against infectious disease. Dectin-2 is selectively expressed by myeloid cells, and upon ligation, mediates enhanced phagocytosis, antigen processing and presentation, and proinflammatory cytokine production. Given these properties, we evaluated the therapeutic potential of targeting Dectin-2 to reprogram TAMs using natural ligands as well as our novel agonistic antibodies. We show that Dectin-2 agonists can convert TAMs into immunostimulatory cells in vitro and in vivo, and drive robust anti-tumor immunity. Dectin-2 gene expression is minimal in normal human tissues but elevated across many tumor types, including breast, colon, lung, ovarian, and kidney cancers. We found that Dectin-2 is strongly expressed by macrophages differentiated in vitro and on primary TAMs from various solid tumors. Treatment of tumor-bearing mice with mannan, a natural Dectin-2 ligand derived from S. cerevisiae, mediated tumor regression in multiple syngeneic tumor models, with high rates of tumor clearance in the MB49 bladder cancer model. These effects were Dectin-2 dependent, as efficacy was not observed when a Dectin-2-blocking antibody was co-administered or in knockout mice lacking Dectin-2 signaling components. Depletion of either macrophages or CD8+ T cells impaired efficacy, suggesting that Dectin-2-stimulated TAMs augment anti-tumor CD8+ T cell responses. Based on these data, we developed novel Dectin-2-targeted agonist antibodies capable of activating both in vitro-generated and primary human TAMs to produce an array of proinflammatory cytokines and chemokines akin to tumor-destructive “M1” macrophages. Furthermore, systemically administered Dectin-2 agonist antibodies activated TAMs and mediated anti-tumor effects in immunodeficient mice engrafted with human CD34+ HSCs. The data presented demonstrate the therapeutic potential of Dectin-2 agonist antibodies as a novel pan-cancer approach for myeloid cell-directed tumor immunotherapy. Citation Format: Justin A. Kenkel, Po Y. Ho, Karla A. Henning, Cindy L. Kreder, Jess L. Nolin, Sameera Kongara, Steven J. Chapin, Amreen Husain, Marcin Kowanetz, Edgar G. Engleman, Michael N. Alonso, David Dornan, Shelley E. Ackerman. Dectin-2 agonist antibodies reprogram tumor-associated macrophages to drive anti-tumor immunity [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 2883.

Abstract 4211: Minocycline-based metronomic therapy as a novel approach to enhance anti-tumor immune responses and reduce tumor burden in mouse models of colorectal cancer

Abstract Despite the availability of numerous new therapies including immune checkpoint inhibitors, patients with colon cancer are faced with limited treatment options. We recently demonstrated that low metronomic dose of PARP inhibitor synergizes with anti-PD-1 immunotherapy, in part, by modulating the suppressive function of MDSCs via a reduction in Arg1 and iNOS but independently of DNA damage and STING (PMC7839867). The non-antibiotic properties of minocycline (Mc) include its ability to moderately inhibit PARP. We took advantage of Mc as a weak PARP inhibitor to explore its potential on tumorigenesis and immune suppression in colon cancer models. In this study, we show that Mc significantly reduced Arg1 and iNOS in bone marrow (BM)-derived MDSCs both at the mRNA and protein levels. Paradoxically, such strong effect did not culminate in a substantial modulation of MDSCs suppressive function as assessed by co-cultured anti-CD3/CD28-stimulated CFSE-labeled T cells proliferation. However and similarly to PARP inhibitors, Mc increased PD-L1 expression in several cell types. We thus speculated that the antibiotic might enhance anti-cancer immunotherapy and explored such potential in a MC-38 and CT-26 cell-based mouse models of colon cancer representing the MSI and MSS traits of the disease. Treatment of tumor-bearing mice with low metronomic doses of Mc (0.5 or 5mkg) promoted a substantial reduction in tumor size in the MC-38 cell-based model (MSI) without an apparent superiority of any of the two doses. Surprisingly, neither dose of Mc exerted appreciable effects on tumor size in the CT-26 cell-based model (MSS). The anti-tumor effect of Mc in the MSI model was associated with a significant reduction in intratumoral monocytic-MDSC population. While MDSCs harvested from tumors of control mice re-infiltrated ex vivo generated MC-38 cell-based organoids, MDSCs harvested from tumors of Mc-treated mice failed to re-infiltrate the organoids. This suggests that Mc may interfere with MDSCs intratumorally infiltration. The Mc concentrations used in this study exerted little to no effect on bacterial growth suggesting that the observed effects were independent of the antibiotic properties of the drug. Unlike the effects observed in vitro, Mc treatment reduced intratumoral PD-L1 while increasing IFNγ. The discrepancy may be associated with intratumoral stress condition as assessed by Chop protein levels and reproduced in vitro by serum starvation. Although the metronomic Mc dose and anti-PD1 administration caused a significant reduction in tumor size individually, the combination therapy promoted a 66.7% curing of the tumors. Our results propose repurposing Mc at metronomic doses as a new approach to enhance efficacy of immunotherapy for patients with MSI colon cancer. Citation Format: Salome V. Ibba, Hanh H. Luu, Mohamed Ghonim, Cecilia Vittori, Matthew J. Dean, Giulia Monticone, Francesca Peruzzi, Augusto Ochoa, Lucio Miele, Hamid A. Boulares. Minocycline-based metronomic therapy as a novel approach to enhance anti-tumor immune responses and reduce tumor burden in mouse models of colorectal cancer [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 4211.

Abstract 2053: Phenotypic and functional analyses of response of lung tumor-associated myeloid cells to systemic oncolytic virotherapy

Abstract Introduction: Oncolytic virotherapy represents a promising cancer treatment modality. One of the appeals of oncolytic virotherapy approaches is the capacity of viruses to change tumor microenvironment, frequently interpreted as de-polarization of tumor-associated macrophages (TAMs) from highly pro-tumorigenic to less pro-tumorigenic phenotypes. The goal of this study was to analyze how systemic therapy with oncolytic adenovirus AVID-317 modulates complexity, phenotypes, and pro-tumorigenic function of tumor-associated myeloid cells in a mouse model of orthotopic metastatic lung cancer. Methods: We utilized single cell RNAseq and a 37-marker mass-cytometry (Cy-TOF) analyses to explore phenotypic and functional states of distinct myeloid cell populations in the lungs of tumor-bearing mice before and after virotherapy. We further performed correlation analyses to reveal functional contribution of each identified myeloid cell population to tumor control or tumor progression after virotherapy on a large groups of mice treated with the virus (N=43) or buffer (N=23). Summary of the results: Our phenotypic immunoprofiling identified 13 discrete TAM and DCs cell populations and 5 discrete populations of granulocytic cells, which were present in the lungs of lung tumor-bearing mice prior to virotherapy treatment. We found that in all TAM populations, the expression of CD274, ARG1, IL-10, and surprisingly, CD127 correlated with a higher tumor burden. In 7 out of 13 TAM populations, virotherapy further increased the expression of these pro-tumorigenic markers. Moreover, virotherapy led to a significant increase in the abundance of TAMs that correlated with tumor burden increase. In addition, in response to virotherapy, TAMs increased the expression of pro-tumorigenic growth factors. These data indicate the virotherapy indeed activated TAMs but did not trigger their de-polarization to lesser pro-tumorigenic phenotypes. To find whether virus-induced tumor cell death may drive TAM activation, we next treated tumor-bearing mice with a UV-inactivated AVID-317 virus that lacks the capacity of killing tumor cells. Our analyses confirmed that virus-mediated tumor cells death but not the exposure of TAMs to the virus drives TAM activation. Conclusion: Our analyses showed that oncolytic virotherapy activates TAMs, which in response produce pro-tumorigenic growth factors that may promote tumor progression. Our findings do not support the universal assumption of de-polarization of TAMs to a lesser pro-tumorigenic phenotypes in response to oncolytic virotherapy. The clinical success of oncolytic virotherapy will require careful consideration of the TAM activation in response to virotherapy and finding effective drug combinations to suppress pro-tumorigenic function of myeloid cells prior to administration of viruses with potent anti-tumor activity. Citation Format: Svetlana Atasheva, Jia Yao, Dmitry Shayakhmetov. Phenotypic and functional analyses of response of lung tumor-associated myeloid cells to systemic oncolytic virotherapy [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 2053.

Abstract 5567: hYP218 CAR-T cells targeting a membrane-proximal epitope of mesothelin are highly effective against mesothelin expressing cancers

Abstract Background and Hypothesis: Mesothelin is an attractive target for CAR-T cell therapy since it is highly expressed in many solid tumors but has limited expression on normal human tissues. However, anti-mesothelin CAR-T cells have shown limited efficacy in patients. Since most antibodies to mesothelin bind the membrane distal epitopes we generated antibodies that bind mesothelin in a membrane-proximal region. We hypothesize that CAR-T cells recognizing a membrane proximal epitope on mesothelin will be more efficacious. Study Design: We developed CAR-T cells targeting a membrane-distal (SS1CAR-T) and a membrane-proximal epitope (hYP218 CAR-T) of mesothelin. Using mesothelin-expressing cancer cell lines, anti-tumor efficacy of CAR-T cells was assessed in vitro by measuring direct killing of tumor cells by the CAR-T cells and quantifying cytokine-release by the CAR-T cells. In vivo anti-tumor efficacy of CAR-T was determined in NSG mice using three different solid tumor models of high-mesothelin expression: ovarian cancer (OVCAR-8), pancreatic cancer (KLM-1), and mesothelioma patient-derived xenograft (NCI-Meso63). Blood and tumor infiltration of CAR-T cells in vivo was determined via flow cytometry analysis. Results: Upon co-culture with tumor cells with high mesothelin expression (A431-H9, OVCAR-8, KLM-1, H226, NCI-Meso63, NCI-Meso29), hYP218 CAR-T cells killed cancer cells more efficiently than SS1 CAR-T cells, with a 2-4-fold lower ET50 value (Effector to Target ratio for 50% killing of tumor cells). Additionally, compared to SS1 CAR-T cells, hYP218 CAR-T cells secreted significantly higher level of cytokines (IL-2, TNFα, IFNγ) in a co-culture with tumor cells. In three solid tumor models, single treatment of tumor-bearing mice with hYP218 CAR-T cells lead to improved tumor response and survival compared to SS1 CAR-T, with complete regression of OVCAR-8 and NCI-Meso63 tumors. Compared to SS1 CAR-T cells, higher hYP218 CAR-T cell infiltration was detected both in the blood and tumors isolated from hYP218 CAR-T treated mice seven days post-treatment. Conclusion: We demonstrate that hYP218 CAR-T cells targeting a membrane-proximal epitope of mesothelin are more potent than similar CAR-T cells targeting a membrane distal epitope. Increased accumulation of hYP218 CAR-T cells in the tumor may explain their increased efficacy. These results support its clinical development to treat patients with mesothelin expressing cancers. Citation Format: Sakshi Tomar, Jingli Zhang, Nan Li, Ira Pastan, Mitchell Ho, Raffit Hassan. hYP218 CAR-T cells targeting a membrane-proximal epitope of mesothelin are highly effective against mesothelin expressing cancers [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 5567.

Abstract 3608: Triple checkpoint blockade, but not anti-PD1 alone, enhances the efficacy of engineered adoptive T cell therapy in advanced ovarian cancer

Abstract Background: Less than half of ovarian cancer patients survive five years after diagnosis. This rate has changed little in the last 30 years, highlighting the need for novel therapies. A promising new strategy with the potential to control tumor growth without toxicity to healthy tissues employs immune T cells engineered to target proteins uniquely overexpressed in tumors. Mesothelin (Msln) is overexpressed in high grade serous ovarian cancer, contributes to the malignant and invasive phenotype, and has limited expression in healthy cells, making it a candidate immunotherapy target in these tumors. Methods: The ID8VEGF mouse cell line was used to evaluate if T cells engineered to express a mouse Msln-specific high-affinity T cell receptor (TCRMsln) can kill murine ovarian tumor cells in vitro and in vivo. Tumor-bearing mice were treated with TCRMsln T cells plus anti-PD-1, anti-Tim-3 or anti-Lag-3 checkpoint-blocking antibodies administered alone or in combination, ultimately allowing targeting up to three inhibitory receptors simultaneously. Single cell RNA sequencing was used to profile the impact of combination checkpoint blockade on both the engineered T cells and the tumor microenvironment. Results: In a disseminated ID8 tumor model, adoptively transferred TCRMsln T cells preferentially accumulated within established tumors, delayed ovarian tumor growth, and significantly prolonged mouse survival. However, our data also revealed that elements in the tumor microenvironment (TME) limited engineered T cell persistence and ability to kill cancer cells. Triple checkpoint blockade, but not single- or double-agent treatment, dramatically increased effector cytokine production by intratumoral TCRMsln T cells. Single cell RNA-sequencing revealed gene expression changes in engineered T cells and myeloid cells in the TME consistent with activation and inflammation. Moreover, combining adoptive immunotherapy with triple checkpoint blockade prolonged survival in the cohort of treated tumor-bearing mice, relative to TCRMsln with or without anti-PD1, or double-agent treatments. Conclusions: Inhibitory receptor/ligand interactions within the tumor microenvironment can dramatically reduce T cell function, suggesting tumor cells may increase expression of the ligands for PD-1, Tim-3 and Lag-3 for protection from tumor-infiltrating lymphocytes. In a model of advanced ovarian cancer, triple checkpoint blockade significantly improved the anti-tumor function of transferred engineered T cells and improved outcomes in mice in a setting in which single checkpoint blockade had no significant activity. These results suggest that disrupting multiple inhibitory signaling pathways simultaneously, which can be more safely pursued in a cell intrinsic form through genetic engineering, may be necessary for improved efficacy in patients. Citation Format: Kristin G. Anderson, Yapeng Su, Madison G. Burnett, Breanna M. Bates, Magdalia L. Rodgers Suarez, Susan L. Ruskin, Aesha Vakil, Valentin Voillet, Raphael Gottardo, Philip Greenberg. Triple checkpoint blockade, but not anti-PD1 alone, enhances the efficacy of engineered adoptive T cell therapy in advanced ovarian cancer [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 3608.

Abstract 1299: Immunotherapeutic hcw9218 enhances anti-tumor activity of chemotherapy by facilitating immune-mediated elimination of therapy induced senescent cells

Abstract Advances in immunostimulatory and anti-immunosuppressive therapeutics have revolutionized cancer treatment. Here, we report a novel heterodimeric bifunctional fusion molecule, HCW9218, constructed using our soluble tissue factor (TF)-based scaffold technology comprising extracellular domains of the human transforming growth factor-β (TGF-β) receptor II and a human interleukin (IL)-15/IL-15 receptor α complex. This fusion complex exhibited TGF-β neutralizing activity in vitro and sequestered plasma TGF-β in vivo. Subcutaneous administration of HCW9218 was well tolerated in mice, with a half-life sufficient to provide long lasting biological activity. HCW9218 enhanced metabolic and cytotoxic activities of immune cells (CD8+ T cells, NK cells) and reduced tumor progression in models of chemotherapy induced senescence (TIS, docetaxel for B16F10 tumors and gemcitabine plus nab-paclitaxel for SW1990 tumor models) and non-TIS in vivo. Mechanistically, HCW9218 treatment not only affected the immunosuppressive tumor microenvironment but also enhanced CD8+ T cells and NK cells infiltration and cytotoxicity in the tumors to eliminate TIS cancer cells. Immune-depletion studies showed that HCW9218-activated NK cells played a pivotal role in TIS cancer cell removal. HCW9218 treatment following docetaxel chemotherapy further enhanced efficacy of tumor antigen-specific and anti-PDL-1 antibodies in B16F10 tumor-bearing mice. We also show that HCW9218 treatment lowered senescence associated secretory phenotype (SASP) factors in off-target tissues in chemotherapy treated tumor-bearing mice. Thus, HCW9218 may serve as a novel therapeutic (monotherapy or in combination with chemotherapy) to simultaneously provide immunostimulation and lessen immunosuppression associated with tumors. HCW9218 has been cleared by the U.S. Food and Drug Administration to proceed for evaluation in a first-inhuman Phase 1b clinical trial in patients with advanced pancreatic cancer. Citation Format: Pallavi Chaturvedi, Varghese George, Bai Liu, Niraj Shrestha, Meng Wang, Micheal Dee, Xiaoyun Zhu, Jack Egan, Jin-an Jiao, Catherine Spanoudis, Jilan Xing, Victor Gallo, Christian Echeverri, Lijing You, Lin Kong, Gabriela Muniz, Peter Rhode, Hing Wong. Immunotherapeutic hcw9218 enhances anti-tumor activity of chemotherapy by facilitating immune-mediated elimination of therapy induced senescent cells [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 1299.

Cationic Nanoparticulate System for Codelivery of MicroRNA-424 and Podophyllotoxin as a Multimodal Anticancer Therapy

Because of the complexity of cancer ecosystems, the efficacy of single-agent chemotherapy is limited. Herein, we report the use of cationic nanoparticles (designated PPCNs) generated from a chemically modified form of the chemotherapeutic agent podophyllotoxin (PPT) to deliver both microRNA-424 (miR-424) and PPT to tumor cells, thus combining chemotherapy and gene therapy. We evaluated the optimal loading ratio of miR-424─which targets programmed cell death ligand 1 (PD-L1) mRNA and reduces PD-L1 production, thus promoting the attack of tumor cells by T cells─for effective delivery of miR-424 and PPCNs into nonsmall-cell lung cancer cells (H460). Because miR-424 can reverse chemotherapy resistance, treatment of the tumor cells with the combination of miR-424 and PPT enhanced their sensitivity to PPT. Because miR-424 and the PPCNs regulated PD-L1 production in different ways, the miR-424@PPCN complexes were significantly more efficacious than either miR-424 or PPCNs alone. We also demonstrated that treatment of tumor-bearing mice with these complexes significantly inhibited tumor growth and extended survival. Moreover, additional in vitro experiments revealed that the complexes could remodel the tumor immune microenvironment, relieve immunosuppression, and achieve immune normalization. This novel system for delivering a combination of PPT and miR-424 shows great potential for the multimodal treatment of lung cancer.

Inhibition of D-2HG leads to upregulation of a proinflammatory gene signature in a novel HLA-A2/HLA-DR1 transgenic mouse model of IDH1R132H-expressing glioma

BackgroundLong-term prognosis of WHO grade II, isocitrate dehydrogenase (IDH)-mutated low-grade glioma (LGG) is poor due to high risks of recurrence and malignant transformation into high-grade glioma. Immunotherapy strategies are attractive...

Cancer vaccines used in combination with TCR-like antibody blockade of the Qa-1b/Qdm complex, the ligand for the immune checkpoint NKG2A, induce anti-tumor immunity

Abstract Antagonizing antibodies against immune checkpoint inhibitory molecules (ICI) has gained an excellent reputation in immuno-oncology. Anti CTLA-4 and anti-PD-1 are among the first cancer checkpoint immunotherapies that got FDA approval to treat certain malignancies. However, not everyone can benefit from them due to the insufficient expression of the checkpoint marker on the surface of the tumor cells. CD94/NKG2A receptor is another inhibitory marker known to be expressed by natural killer cells and sub-population of CD8+ T cells. Specific peptides loaded by Qa-1b (mouse) or HLAE (human) on the surface of tumor cells, immune infiltrating lymphocytes, and myeloid cells are NKG2A ligand and are known to have a significant role in modulating anti-tumor effector cell responses. Our group previously developed a single-domain T-cell receptor-like antibody (EXX-1) with specificity for the Qa-1b/Qdm peptide complex, the ligand for the murine NKG2A/CD94 receptor. We have shown that treating tumor-bearing mice with EXX-1 as a single agent blocking antibody can create a modest delay in mice tumor growth and overall survival time. The presented study focused on utilizing EXX-1efficacy as a checkpoint blocking antibody in combination with a cancer vaccine. Our findings indicate that cancer vaccine effectiveness can be markedly enhanced when combined with EXX-1 antibody, leading to tumor-free survival in 35% of the mice compared to control groups. Taken together, our results indicate targeting the NKG2A axis through blocking Qa-1b/Qdm peptide complex is an effective means to treat cancer. Also, using HLAE/Qa-1b blocking antibody can improve the efficacy of the cancer vaccines and lead to better clinical outcomes when combined.

A Sampling of Highlights from the Literature: Article Recommendations from Our Deputy and Senior Editors.

TLS formation can be driven by Hhep-specific Tfh cells (from Fig. 3A of Wennhold et al., Cancer Immunol Res 2021)Intestinal microbiota influence the development of colorectal cancer (CRC) and antitumor immunity. Overacre-Delgoffe et al. show that intestinal colonization with Helicobacter hepaticus (Hhep) reduces tumor burden in a mouse model of CRC. CD4+ T cells, B cells, and NK cells, but not CD8+ T cells, mediate antitumor immunity in the Hhep-colonized mice. Mechanistically, Hhep colonization drives the expansion of CD4+ T follicular helper (Tfh) cells and Hhep-specific Tfh cells promote the development of peritumoral tertiary lymphoid structures (TLS). The data provide new insight into the ways that intestinal microbiota can impact antitumor immunity.Overacre-Delgoffe AE, …, Hand TW. Immunity 2021 Dec 14;54:2812–24.e.4.A B cell–Tfh cell–IL21 axis could be a new therapeutic target for LUAD (by Nephron via Wikimedia Commons)The presence of CD4+ T follicular helper (Tfh) cells and B cells in tumors correlates with improved outcomes, but the functional significance of these cells for antitumor immunity is not well defined. After finding that Tfh cells and germinal center (GC) B cells correlate with improved survival among patients with lung adenocarcinoma (LUAD), Cui et al. show that B-cell recognition of a tumor-expressed neoantigen promotes tumor-specific Tfh- and GC-cell responses in a mouse model of LUAD. IL21 production by tumor-specific Tfh cells drives antitumor CD8+ T-cell responses. The data identify a B cell–Tfh cell–IL21 axis that could provide a new target for developing new cancer immunotherapies.Cui C, …, Joshi NS. Cell 2021 Dec 9;184:6101–18.e13.CAR T cells can be engineered to overcome exhaustion due to chronic antigenic stimulation (from Fig. 1 of Okoye et al., Front Immunol 2017)The efficacy of chimeric antigen receptor (CAR) T cells against solid tumors is limited. Using a model of pancreatic cancer, Good et al. show that chronic stimulation of mesothelin-targeted CAR T cells leads them to become exhausted and undergo a transition into an NK cell–like phenotype. Expression of ID3 and SOX4 are upregulated with exhaustion and associate with the upregulation of NK-cell receptors. Preventing expression of ID3 or SOX4 in CAR T cells can improve antitumor responses and reverse CAR T-cell dysfunction, highlighting a potential strategy to improve the efficacy of such therapy against solid tumors.Good CR, …, June CH. Cell 2021 Dec 9;184:6081–100.e26.TCR signal strength can balance T-cell function and antitumor killing (by Clker-Free-Vector-Images via Wikimedia Commons)TCR signaling strength dictates T-cell responses. By studying the effects of signal strength on antitumor responses, Shakiba et al. show that a high signal strength leads to increased expression of inhibitory receptors, and subsequent loss of effector function and dysregulation. Low TCR signals lead to upregulation of PD-1, but T cells retain their functional status. However, low TCR signals are not sufficient for tumor clearance in vivo. Fine-tuning the TCR signal strength to intermediate to balance T-cell function and antitumor killing improves tumor clearance. The data highlight the importance of TCR signal strength in the efficacy of T cell–based cancer immunotherapies.Shakiba M, …, Schietinger A. J Exp Med 2021 Dec 22;219:e20201966.SOCS1 puts the brakes on CD4+ T-cell proliferation and activity (by Capri23auto via Pixabay)The reasons that CD4+ T cells do not proliferate as extensively as CD8+ T cells after antigen stimulation in vivo are not well characterized. Sutra Del Galy et al. conducted a genome-wide CRISPR screen on mouse CD4+ T cells, identifying Socs1 as a negative regulator of expansion in antigen-experienced CD4+ T cells in vivo. Inactivation of Socs1, which integrates IL2 and IFNγ cytokine signaling, in tumor-specific CD4+ and CD8+ T cells, as well as in human chimeric antigen receptor T cells, results in improved antitumor activity in mouse tumor models. These insights expand our understanding of negative regulators of the T-cell response, which may inform improved design of adoptive T-cell therapies.Sutra Del Galy A, …, Menger L. Sci Immunol 2021 Dec 3;6:eabe8219.Macrophages expressing MARCO accumulate lipids and contribute to tumor immune evasion (by NIAD via Flickr)Tumor-associated macrophages (TAM) can enhance prostate adenocarcinoma growth and immune evasion, but a deeper understanding of the underlying mechanisms is needed. Masetti et al. identify a subset of TAMs within highly aggressive prostate adenocarcinoma tissue samples that exhibit aberrant lipid accumulation, lipid metabolism, and high expression of MARCO. This TAM subset is associated with worse patient outcomes and increased tumor aggressiveness in mice. Treating tumor-bearing mice with a MARCO-blocking antibody reduced tumor growth, and resulted in fewer lipid-loaded and more proinflammatory tumor-infiltrating macrophages. The data highlight an immune pathway that may inform clinical prognosis and the development of future immunotherapies.Masetti M*, Carriero R*, …, Di Mitri D. J Exp Med 2021 Dec 17;219:e20210564.

Relationship between the oxidative status and the tumor growth in transplanted triple-negative 4T1 breast tumor mice after oral administration of rhenium(I)-diselenoether

BackgroundSelective inhibitory effects of rhenium(I)-diselenoether (Re-diSe) were observed in cultured breast malignant cells. They were attributed to a decrease in Reactive Oxygen Species (ROS) production. A concomitant decrease in the production of Transforming Growth Factor-beta (TGFβ1), Insulin Growth Factor 1 (IGF1), and Vascular Endothelial Growth Factor A (VEGFA) by the malignant cells was also observed. AimThe study aimed to investigate the anti-tumor effects of Re-diSe on mice bearing 4T1 breast tumors, an experimental model of triple-negative breast cancer, and correlate them with several biomarkers. Material and methods4T1 mammary breast cancer cells were orthotopically inoculated into syngenic BALB/c Jack mice. Different doses of Re-diSe (1, 10, and 60 mg/kg) were administered orally for 23 consecutive days to assess the efficacy and toxicity. The oxidative status was evaluated by assaying Advanced Oxidative Protein Products (AOPP), and by the dinitrophenylhydrazone (DNPH) test in plasma of healthy mice, non-treated tumor-bearing mice (controls), treated tumor-bearing mice, and tumors in all tumor-bearing mice. Tumor necrosis factor (TNFα), VEGFA, VEGFB, TGFβ1, Interferon, and selenoprotein P (selenoP) were selected as biomarkers. ResultsDoses of 1 and 10 mg/kg did not affect the tumor weights. There was a significant increase in the tumor weights in mice treated with the maximum dose of 60 mg/kg, concomitantly with a significant decrease in AOPP, TNFα, and TGFβ1 in the tumors. SelenoP concentrations increased in the plasma but not in the tumors. ConclusionWe did not confirm the anti-tumor activity of the Re-diSe compound in this experiment. However, the transplantation of the tumor cells did not induce an expected pro-oxidative status without any increase of the oxidative biomarkers in the plasma of controls compared to healthy mice. This condition could be essential to evaluate the effect of an antioxidant drug. The choice of the experimental model will be primordial to assess the effects of the Re-diSe compound in further studies.

(S)-(-)-N-[2-(3-Hydroxy-2-oxo-2,3-dihydro-1H-indol-3-yl)-ethyl]-acetamide inhibits colon cancer growth via the STAT1 pathway

(S)-(-)-N-[2-(3-Hydroxy-2-oxo-2,3-dihydro-1H-indol-3-yl)-ethyl]-acetamide (EA), a new compound isolated from the plant Selaginella pulvinata, has been identified to possess anti-proliferative effects against SK-mel-110 cells in vitro via upregulating the expression of inhibitor of growth family member 4. The present study aimed to investigate whether EA could exert antitumor effects against colon cancer. The results revealed that EA exerted anti-proliferative effects against a broad spectrum of different types of tumor and could induce apoptosis of colon cancer cells via the signal transducer and activator of transcription 1 pathway. It was demonstrated that treating tumor-bearing mice with EA significantly inhibited colon tumor growth in a xenograft tumor model and syngeneic tumor model. Furthermore, EA treatment reshaped the tumor microenvironment by decreasing myeloid-derived suppressor cell accumulation and increasing the tumor infiltrating lymphocytes. The results from the present study suggest that EA could be developed as a potential antitumor drug against colon cancer.

Abstract PO-072: Inhibiting vasoactive intestinal peptide receptor signaling elicits T cell dependent anti-tumor response of pancreatic ductal adenocarcinoma to immune checkpoint therapy

Abstract Pancreatic ductal adenocarcinoma (PDAC) is unresponsive to immune checkpoint therapy largely due to a paucity of T cells within the tumor microenvironment (TME) and abundant immunosuppressive signaling pathways. In this study we show that human PDAC tumors over-express vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide that suppresses T cell effector properties and promotes the generation of regulatory T cells (Tregs). Therefore, we treated tumor-bearing mice with VIP receptor peptide antagonists and measured T cell homing, activation, and anti-tumor responses in preclinical murine models of PDAC. Pharmacological inhibition of VIP receptor (VIP-R) signaling using daily subcutaneous injections of peptide antagonists had no discernable toxicity in healthy and tumor-bearing mice. Treatment with VIP-R antagonists in combination with anti-PD-1 checkpoint blockade significantly decreased tumor burden, and improved survival in subcutaneous and orthotopic murine PDAC models. Combination therapy significantly enhanced T cell activation and proliferation and decreased frequencies of Tregs within the TME. Anti-tumor responses were T cell dependent, as the combination therapy failed to improve survival in CD4 or CD8 deficient mice using knock-out strains and antibody depletion. Furthermore, combination therapy significantly increased frequencies of tumor specific T cells (measured with a tetramer reagent) and provided protective immunity against tumor rechallenge. Combination therapy led to significant increases in the infiltration of adoptively transferred GFP+ T cells into PDAC tumors and decreased CXCR4 expression levels on T cells. Encouragingly, peptide-based VIP-R antagonists enhanced the in vitro activation of human T cells isolated from peripheral blood of PDAC patients. Human T cells cultured with VIP-R antagonists had increased proliferation, activation, and decreased proportions of T regs and exhausted T cells co-expressing PD-1, Tim-3 and Lag-3. Taken together, our findings show that VIP is a targetable mechanism of immune escape in PDAC. Inhibiting VIP receptor signaling improves T cell effector properties and synergistically improves anti-tumor responses to checkpoint inhibitors in mouse PDAC models. Additionally, as the VIP sequence is identical between human and mice, and since VIP-R antagonists have similar effects on human and murine T cells in culture, clinical translation is highly feasible. Citation Format: Sruthi Ravindranathan, Passang Tenzin, Jian Ming Li, Rohan Dhamsania, Michael Ware, Mohammad Zaidi, Shuhua Wang, Jingru Zhu, Maria Cardenas, Yuan Liu, Gaurav Joshi, Sanjeev Gumber, Brian Robinson, Anish Sen-Majumdar, Shanmuganathan Chandrakasan, Haydn Kissick, Alan Frey, Susan Thomas, Bassel El-Rayes, Gregory Lesinski, Edmund K. Waller. Inhibiting vasoactive intestinal peptide receptor signaling elicits T cell dependent anti-tumor response of pancreatic ductal adenocarcinoma to immune checkpoint therapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-072.

Inhibition of tumor-associated macrophages by trabectedin improves the antitumor adaptive immunity in response to anti-PD-1 therapy.

A considerable proportion of cancer patients are resistant or only partially responsive to immune checkpoint blockade immunotherapy. Tumor‐Associated Macrophages (TAMs) infiltrating the tumor stroma suppress the adaptive immune responses and, hence, promote tumor immune evasion. Depletion of TAMs or modulation of their protumoral functions is actively pursued, with the purpose of relieving this state of immunesuppression. We previously reported that trabectedin, a registered antitumor compound, selectively reduces monocytes and TAMs in treated tumors. However, its putative effects on the adaptive immunity are still unclear. In this study, we investigated whether treatment of tumor‐bearing mice with trabectedin modulates the presence and functional activity of T‐lymphocytes.In treated tumors, there was a significant upregulation of T cell‐associated genes, including CD3, CD8, perforin, granzyme B, and IFN‐responsive genes (MX1, CXCL10, and PD‐1), indicating that T lymphocytes were activated after treatment. Notably, the mRNA levels of the Pdcd1 gene, coding for PD‐1, were strongly increased. Using a fibrosarcoma model poorly responsive to PD‐1‐immunotherapy, treatment with trabectedin prior to anti‐PD‐1 resulted in improved antitumor efficacy. In conclusion, pretreatment with trabectedin enhances the therapeutic response to checkpoint inhibitor‐based immunotherapy. These findings provide a good rational for the combination of trabectedin with immunotherapy regimens.

Anti-tumor activity of nanoliposomes containing crude extract of saffron in mice bearing C26 colon carcinoma

Objective(s): Saffron, the dehydrated stigma of the Crocus sativus L. flower, has been reputed as an effective anticancer and chemopreventive agent in cancer therapy. This study aimed to design PEGylated nanoliposomes containing crude extract of saffron for the treatment of cancer.Materials and Methods: Various PEGylated nanoliposomes containing 25 mg/ml aqueous extract of saffron were prepared using the thin lipid film method. The characterization of liposomes was indicated by their size, in vitro cytotoxicity, and in vivo therapeutic efficacy against C26 tumor-bearing mice. Results: By increasing cholesterol levels, the IC50 values of the formulations increased. Liposome characterization illustrated the properties of formulation of choice, as follows: Z-average size: 73.7 ± 1.3 nm; PDI: 0.103 ± 0.035; zeta potential: -20.8 mV ± 3.7; % encapsulation: 91 ± 0.059, % release after 168 hours in 30% FBS: 16.26 ± 0.01.Conclusion: Treating tumor-bearing mice with the selected saffron liposomes indicated that, for the first time, the i.v. injection of nano-liposomal saffron at a dose of 300 mg/kg significantly increased the anti-tumor property compared to the negative control group, while no significant difference was observed compared with aqueous extract of saffron. Hence, to achieve an optimal formulation for human use, the formulation merits further study.