Suppressive Tumor Microenvironment Research Articles

Abstract 5679: Tetra-specific antibody GNC-035: guidance and navigation control (GNC) molecule development for treatment of ROR1+ malignancies

Abstract Cancer-intrinsic immune escape mechanisms and immune cell suppression can progressively diminish the curative potential of currently available T cell-based therapies. Barriers to successful T cell checkpoint therapies may be addressed by redirection of T cells toward tumor antigens using T cell engagers that function independently of MHC presented T cell epitopes. Here we demonstrate that an octavalent, tetraspecific Guidance and Navigation Control (GNC) antibody, GNC-035, binds to ROR1, CD3, PD-L1, and 4-1BB and mediates redirected T cell cytolysis of human solid tumor and leukemia and lymphoma cell lines in a ROR1 specific manner. Experiments using GNC-035 to redirect T cell cytotoxicity toward ROR1+ cancer cell targets show the T cells in PBMC are highly functionalized by pre-exposure to GNC-035. This pre-exposure of PBMC to GNC-035 results in greater tumor cell killing efficacy compared to concurrent exposure of tumor cells in the presence of T cell effectors. This result suggests that the systemic delivery of GNC-035 can condition the T cell compartment to increase the therapeutic impact of T cells migrating to solid tumors, with or without preexisting infiltrating T cells. This beneficial conditioning of T cells by pre-exposure to GNC-035 is not observed with pre-exposure to CD3xROR1 bi-specific T cell engager controls. To evaluate the potential for GNC-035 to mediate cytokine release syndrome, the molecule is evaluated in soluble formats in the presence of PBMC and the ROR1+ A549 cancer cells, or HUVEC cells. Under these conditions, the cytolysis of A549 target cells is detectable after exposure to GNC-035 at 100 fM concentrations as well as the release of IFN-γ and certain other inflammatory cytokines at 24 or 48 hours post-treatment. However, consistent with Blinatumomab treatment, PBMC exposed to soluble GNC-035 for 24 or 48 hours on a monolayer of HUVEC cells, produced significantly greater amounts of IFN-γ and IL-6 at concentrations greater than 10 pM. These results indicate GNC-035 has a therapeutic window of activity that is ROR1 dependent, spanning cytolytic activity, and IFN-γ release without a production of IL-6 and which is wider than that indicated by Blinatumomab in PBMC. Collectively, the GNC-035 represents a class of multi-specific and multi-modal immune cell engagers with potential to mediate ROR1+ cancer regression, overcome TCR-based immune escape and reverse T cell immune suppression in tumor microenvironment. The clinical phase I-b study of GNC-035 is under way in breast cancer and hematologic cancers and the available data exhibit strong signals of efficacy with acceptable tolerability. Citation Format: Jahan Salar Khalili, Sa Xiao, Yi Zhu. Tetra-specific antibody GNC-035: guidance and navigation control (GNC) molecule development for treatment of ROR1+ malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5679.

Abstract 5111: VentX-modulated tumor associated macrophages revert immune suppression in tumor microenvironment and promote efficacy of gemcitabine against pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy with median survival of less than one year and overall 5-year survival less than 5%. Gemcitabine has been the cornerstone in PDA treatment, yet its efficacy remains limited. Over the past decades, extensive investigations suggested a role of tumor microenvironment (TME) in rendering PDA resistance to gemcitabine, however, its underlying mechanism remained largely unknown. Cellular profiling has revealed the macrophages as a major immune cell component of PDA-TME. Our recent study showed that the expression of the homeobox protein VentX, a master regulator of macrophage plasticity, is significantly down-regulated in tumor associated macrophages (TAMs) of PDA. We demonstrated that restoration of VentX expression in PDA-TAMs polarizes the TAMs from a pro-tumor M2-like phenotype to an anti-tumor M1-like phenotype. We showed further that VentX-regulated-TAMs (VentX-TAMs) revert immune suppression of PDA-TME by inhibiting CD4 Treg differentiation and by promoting CD8 proliferation and activation. Using a newly developed tumor immune microenvironment-enabling model system (TIME-EMS), we showed that VentX-TAMs drastically promote efficacy of gemcitabine against PDA for about 4-fold but not cytotoxicity on normal pancreatic tissue. As such, our data suggested a function of VentX-TAMs to promote chemosensitivity of gemcitabine and to improve PDA prognosis. Citation Format: Joanna Le, Hong Gao, Yi Le, William Richards, Scott Radig, Ronald Bleday, Thomas Clancy, Zhenglun Zhu. VentX-modulated tumor associated macrophages revert immune suppression in tumor microenvironment and promote efficacy of gemcitabine against pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5111.

Abstract 4124: Targeting the clock pathway to modulate immune response in MSI-high colorectal cancer (CRC): evidence from a preclinical in vivo model

Abstract Background. Immune checkpoint inhibitors have substantial clinical success in MSI-H/dMMR metastatic CRC. However, only a subset of patients exhibits durable responses, emphasizing the importance of identifying mechanism of innate and acquired resistance. The relation between the circadian clock and the immune system is well established. We used a syngeneic mouse model of MSI-H CRC to investigate whether a targeted enhancement of clock repressor proteins cryptochrome 1/2 (CRY 1/2) may affect tumor growth and response to anti-PD1 (aPD1). Methods. 1 × 106 MC38 cells were subcutaneously implanted into the right flank of 8 weeks old C57BL/6 mice. Mice were randomized into 1 control and 3 treatment arms (10 mice/group): A. CRY stabilizer SHP656(10 mg/kg PO, 5 days/week); B. aPD1 (100 μg/mouse IP, twice/week administered for 2 weeks); C. combination SHP656 + aPD1. Tumors from 4 mice/group were processed for tumor infiltration studies. Remaining 6 mice/group were kept for 3 weeks on maintenance aPD1 (twice/week) for survival studies. Treatment response was measured by tumor growth and survival. Treatment effects on immune cell infiltration in the tumor microenvironment (TME), functional myeloid derived suppressor cells (MDSCs) and T cells markers were investigated via flow cytometry. Results. Single agent SHP656 significantly improved survival when compared with vehicle treated mice. Similarly, combination treatment with SHP656 + aPD1 improved survival, however no additional benefit was observed over treatment with aPD1 alone. All treatment groups had a response rate of 67% (4/6 mice/treatment group showed 2-fold decrease in tumor volume 24 days after tumor challenge vs vehicle). In responders, SHP656 as a single agent or in combination with aPD1 were more effective in suppressing tumor growth as compared to aPD1 alone. Flow cytometry of 14 tumor samples demonstrated that monocytic-MDSC infiltration was significantly decreased in the combination therapy and aPD1 alone treatment arms (p = .016) as compared to vehicle suggestive of a less suppressive TME. T cell analysis suggested a decrease in intratumoral exhausted CD8+ T cells (TIM3−PD-1+, p = .02) in SHP656 + aPD1 vs vehicle, while infiltration of CD4+ T cells and T regulatory cells were not significantly affected by any treatment. Nanostring profiling on treated tumors also confirmed immune activation in responders. Conclusions. Our results show for the first time that targeting the clock pathway through modulation of CRY affects TME and tumor progression in in vivo models of MSI-H CRC. A decrease in infiltration of mMDSCs and exhausted CD8+ T cells seems to play a critical role in combination treatment efficacy but needs to be studied further. These results, suggests that pharmacological targeting of the circadian machinery holds great potential and could significantly impact the efficacy of immunotherapy in metastatic CRC. Citation Format: Shivani Soni, Francesca Battaglin, Sofi Castanon, Jae Ho Lo, Goar Smbatyan, Priscilla Chan, Meng Qu, Priya Jayachandran, Hiroyuki Arai, Natsuko Kawanishi, Wu Zhang, Steve A. Kay, Heinz Josef Lenz, Evanthia T. Torres. Targeting the clock pathway to modulate immune response in MSI-high colorectal cancer (CRC): evidence from a preclinical in vivo model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4124.

Abstract 1791: Next generation UltraCAR-T® cells with intrinsic checkpoint inhibition and overnight manufacturing overcome suppressive tumor microenvironment leading to sustained antitumor activity

Abstract The UltraCAR-T® platform, with decentralized overnight manufacturing, has shifted the autologous CAR-T manufacturing paradigm. This disruptive approach has been validated in clinical and pre-clinical studies for both hematologic and solid tumors. UltraCAR-T cells, manufactured using an overnight process with minimal ex vivo manipulation of autologous T cells. UltraCAR-T cells are engineered to co-express CAR, membrane bound IL-15, and kill switch genes using non-viral gene transfer via high-throughput UltraPorator™ system. Multigenic design and overnight manufacturing renders UltraCAR-T cells with enhanced in vivo expansion and persistence for durable antitumor activity. Chronic antigen stimulation from the tumor can lead to CAR T having an exhausted phenotype contributing to treatment failures. Next generation UltraCAR-T cells also incorporate a novel cell intrinsic blockade of PD-1, superseding the need for combination therapy with checkpoint inhibitors and mitigate classic T cell exhaustion that occurs from chronic stimulation, thereby expanding the therapeutic window for efficacy. Using mesothelin (MSLN) as an exemplar from the CAR-target library, we developed a robust in vitro and in vivo model of continuous tumor antigen exposure to evaluate antitumor activity of the UltraCAR-T cells. MSLN UltraCAR-T cells were generated with and without intrinsic PD-1 blockade. In vitro, UltraCAR-T cells with intrinsic PD-1 blockade demonstrated enhanced inflammatory cytokine expression and cytotoxicity at low effector:target ratios. In long-term co-culture assays with recurring challenge of MSLN+ PD-L1+ tumor cells every 2-3 days, UltraCAR-T cells with intrinsic PD-1 blockade showed sustained potent cytotoxicity which was superior to CAR-T cells lacking PD-1 blockade and in combination with αPD-1 antibody. Intrinsic PD-1 blockade markedly enhanced polyfunctionality of UltraCAR-T cells in the presence of MSLN+ PD-L1+ tumor. In xenograft solid tumor model, a single administration of MSLN UltraCAR-T cells manufactured using UltraPorator demonstrated significant antitumor efficacy. Blood analysis showed sustained downregulation of PD-1, robust antigen specific expansion and durable persistence with central memory/stem-cell memory as the dominant phenotype of UltraCAR-T in vivo. Rechallenging the mice, who became tumor-free after a single UltraCAR-T infusion, with tumor for a second time to simulate tumor relapse led to the significant reduction in tumor burden without additional UltraCAR-T treatment. Collectively, these pre-clinical data highlight the improved efficacy of intrinsic PD-1 blockade in next generation UltraCAR-T cells using non-viral gene delivery and an established rapid, decentralized manufacturing process. Citation Format: Giorgio Zenere, Carol Poortman, Afreen Sayed, Devi Gunasekera, Cheryl Bolinger, Pooja Chauhan, Jacques Plummer, Shamim Ahmad, Rutul R. Shah, Helen Sabzevari, Douglas E. Brough, Tim Chan. Next generation UltraCAR-T® cells with intrinsic checkpoint inhibition and overnight manufacturing overcome suppressive tumor microenvironment leading to sustained antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1791.

Abstract 1259: Disturbing the Siglec-Sialoglycan axis to target myeloid- derived suppressor cells in the tumor microenvironment

Abstract Background: Overexpression of sialic acids on glycans, called hypersialylation is a common alteration in cancer that drives immune evasion via interaction with Sialic acid-binding immunoglobulin-like lectin (Siglec) immunoregulatory receptors on tumor-infiltrating immune cells. Myeloid derived suppressor cells (MDSCs) generated by cancer-induced aberrant myelopoiesis are highly immunosuppressive cells inhibiting immune cells via direct interaction or secretion of suppressive cytokines. Targeting the complex suppressive tumor microenvironment (TME) remains a challenge. Methods: Siglec expression of murine and human MDSCs in healthy conditions and tumor setting was assessed by flow cytometry. Functional analysis of Siglec-E knockout on MDSCs in mice was evaluated using SigE-LysMCre mice and suppressive capacity was tested in vitro in combination with SiglecE blocking and cleavage of Sialoglycans using Sialidase. Results were confirmed in the human setting using an in vitro assay to generate MDSC-like cells including RNA-Sequencing and a MDSC suppression assay with cancer-derived MDSCs. Results: MDSCs express multiple inhibitory Siglec receptors in humans and mice. Knockout of SiglecE in the myeloid compartment in vivo resulted in prolonged survival and increased T cell infiltration compared to litter mates expressing Siglec-E. Of note, desialylation of MDSCs and Siglec-E blocking in vitro reduced MDSCs suppressive function against T cells. Similarly, reducing Sialoglycans by Sialidase in human MDSC generated in vitro resulted in increased suppressive capacity and downregulation of functional MDSC markers on RNA level. Findings were validated with lung cancer patient derived MDSCs. Conclusion: Our results provide first insights into the importance of the Siglec-Sialoglycans axis to modulate MDSCs in the TME. Targeting glycosylation of MDSCs with Sialidase reduces their suppressive capacity making it a powerful approach to improve cancer immunotherapy. Further studies are needed to reveal the underlying mechanisms. Citation Format: Ronja Wieboldt, Andreas Zingg, Emanuele Carlini, Anastasiya Börsch, Heinz Läubli, Natalia Rodrigues Manutano. Disturbing the Siglec-Sialoglycan axis to target myeloid- derived suppressor cells in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1259.

Abstract 899: Deciphering the benefits of variable delivery routes and molecular armoring to enhance efficacy of MUC1-CAR T-cells in targeting triple-negative breast cancer

Abstract As immune-therapies continue to develop for solid tumors, the tumor microenviroment (TME) poses many challenges that CAR T-cells need to overcome for efficient tumor cell clearance. Tumor immune evasion mechanisms, presence of immunosuppressive factors and cellular resistance to tumor infiltration are key mechanisms that the TME uses to reduce efficacy of T-cells against the tumor. Therapeutic options for triple negative breast cancer (TNBC) remain limited to date despite it being the most aggressive subtype of breast cancers and carrying the poorest prognosis. Tumor-associated MUC1 antigen is overexpressed in a large number of TNBC patients offering an effective discriminatory target for CAR T-cell therapy. Here, we present innovative strategies exploring alternative delivery routes and purposeful armoring of CAR T-cells to boost efficiency while preserving safety. To maintain anti-tumor CAR T-cell cytotoxic and proliferation in the hostile TME of solid tumors, we developed state-of-the-art multiplexed gene editing using high precision TALEN® technology. With that aim, we armored allogeneic CAR T-cells with specific attributes to release immune inflammatory cytokines and protect from inhibitory effects of the TGFB1 and PD1 pathways. A combination of strategies using allogeneic CAR T-cells with diverse attributes were evaluated against TNBC tumors in various in vivo pre-clinical models. Local CAR T-delivery resulted in effective control of tumor growth and robust antigen specific recognition. Furthermore, we assessed how the delivery route contributes to CAR-T cell distribution. CAR T-cell activity could also be increased systemically upon tumor-specific release of immunostimulatory cytokines without compromising safety. Our results demonstrate not only superior activity of armored MUC1-CAR T-cells resulting in tumor clearance but also in the recovery of normal glands. Thus, innovative strategies can be used to allow CAR T-cell efficiency in the hostile tumor microenvironment while preserving safety. Overall, our pre-clinical results underline the capability of armored allogenic MUC1-CAR T-cells to excel in the immune suppressive tumor micro-environment, suggesting that allogenic MUC1-CAR T-cell therapy could be an effective option in treating relapsed/refractory TNBC patients with limited therapeutic options. Citation Format: Piril Erler, Hana Cho, Jordan Skinner, Laurent Poirot, Beatriz Aranda-Orgilles. Deciphering the benefits of variable delivery routes and molecular armoring to enhance efficacy of MUC1-CAR T-cells in targeting triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 899.

Abstract 3246: Targeting Syk reprograms tumor-associated macrophages and enhances responses to immune checkpoint blockade and radiation therapy in high-risk neuroblastoma

Abstract Neuroblastoma (NB) is the most common extracranial solid malignancy in children. As leading regulators of inflammation and tumor progression, tumor associated macrophages (TAMs) have gained major interest as immunotherapeutic targets in NB. Hence targeting or “re-educating” tumorigenic macrophages towards an immunostimulatory phenotype might improve responses to immunotherapy in this childhood cancer. Spleen tyrosine kinase (Syk) has recently been identified by our group as a novel immune-oncology target. We found that Syk is abundantly present in TAMs infiltrated in MYCN and non-MYCN amplified neuroblastoma tumors. Furthermore, genetic deletion or pharmacological inhibition of Syk markedly impaired neuroblastoma tumor growth. This effect was facilitated by macrophages that became immunogenic in the absence of Syk, skewing the suppressive tumor microenvironment towards immunostimulation and activating antitumor immune responses. Moreover, combining Syk inhibitor, R788 along with anti-PDL1 mAb and radiation provided a synergistic effect leading to complete tumor regression and durable anti-tumor immunity in aggressive MYCN-and non-MYCN-driven murine neuroblastoma tumor models. Collectively, our findings demonstrate the importance of Syk inhibitor, R788, in enhancing the anti-tumor immune responses in NB and support the clinical evaluation of R788 either alone or together with radiation and immune check point inhibitors as a potential treatment strategy for NB. Citation Format: Deepak Rohila, In Hwan Park, Timothy Pham, Riley Jones, Pablo Tamayo, Alice Yu, Andrew Sharabi, Shweta Joshi. Targeting Syk reprograms tumor-associated macrophages and enhances responses to immune checkpoint blockade and radiation therapy in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3246.

Abstract 2948: A humanized therapeutic antibody against S100A4 as a novel immune modulator and an inhibitor of cancer metastasis

Abstract Widespread cancer metastasis still contributes to ~ 90% of cancer-related deaths, and limited progress has been made in treating the metastatic disease. S100A4 is a highly conserved protein and has been demonstrated to promote cancer metastasis and stemness in multiple cancer types. Furthermore, our recent single cell RNA-sequencing analysis of cancer and stromal cells in human gliomas showed that S100A4 is expressed in both cancer and immune cells to promotes immune suppressive tumor microenvironment. Significantly deleting S100A4 function in stromal cells alone was sufficient to reverse the immune phenotypes of glioma associated T cells and myeloid cells, and resulted in significantly extend survival of two different mouse glioma models. Consistently, higher S100A4 expression is also associated with poor outcomes in many cancer types. Together these observations suggest that S100A4 is a promising therapeutic target for multiple cancer types, and we developed and characterized a blocking antibody against human S100A4. We have screened 114 monoclonal antibodies against S100A4 using S100A4-induced chicken neurite outgrowth ex vivo as a functional assay. Seven of the 114 monoclonals showed significant suppressive activity ex vivo and were selected for further analysis for their in vivo activity. We chose two well-characterized breast cancer metastasis models for in vivo screening: MMTV-PyMT spontaneous and 4T1 orthotopic transplantation tumor models. One of the clones, 4-11, showed the most consistent and dramatic lung metastasis suppressive activity in both models. To identify the potential mechanisms of action since the blocking antibody is anticipated to only affect soluble S100A4 function, we focused on potential immune modulatory effect, based on our own study and those of others. We performed flow cytometry to analyze the immune phenotypes in blood, bone marrow, spleen, breast tumor, and lung metastases. In S100A4-11 treated mice, circulating monocytes (CD11b+Ly6gloLy6chi) and tumor-associated CD163+ macrophage frequencies were significantly decreased in both models. In addition, circulating CD4+ T cells and neutrophils (CD11b+Ly6g+Ly6cint) frequencies were also decreased in the 4T1 model. Others have shown that myeloid-derived suppressive cells (MDSC: CD11b+, Ly6gloLy6chi,IAIE-) play an important role in cancer metastasis. Our result suggests that soluble S100A4 protein promotes MDSC polarization or survival in the tumor, peripheral blood, and metastatic lung tissues. These findings strongly suggest that S100A4-11 could be used to block metastasis. We are currently testing the humanized S1004-11 antibody in preparation for clinical studies with this novel immune modulator. Citation Format: Jia-Shiun Leu, Hui Deng, Han Nhat Tran, Jose Maria Salazar, Jose A. Maldonado, Carlo D. Cristobal, Xiong Wei, Hyun-Kyoung Lee, Zhiqiang An, Ningyan Zhang, Kyuson Yun. A humanized therapeutic antibody against S100A4 as a novel immune modulator and an inhibitor of cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2948.

Abstract 1158: Inosine endows CAR T cells with features of increased stemness and anti-tumor potency

Abstract Chimeric antigen receptors (CARs) are synthetic receptors that link an extracellular tumor specific domain to intracellular signaling domains. Despite of remarkable results against refractory B cell malignancies, intense effort is underway to augment the potency of CAR T cells in order to overcome the suppressive tumor microenvironment, which is associated with T cell exhaustion. Adenosine is a major mediator of immune suppression. Ectoenzyme CD39 plays a central role in the generation of adenosine by catalyzing the metabolism of ATP into ADP/AMP. Then CD73 subsequently metabolizes ADP/AMP into adenosine which mediates immune suppression through adenosine associated receptor signaling. CD39 is also expressed by exhausted CD8+ and tumor reactive T cells within the tumor microenvironment, where it is associated with tumor progression, but it remains unclear whether exhausted and/or tumor reactive CD39+CD8+ T cells mediate immune suppression via the purigenic pathway. We developed a high affinity version of the disialoganglioside (GD2)-targeting chimeric antigen receptor (HA-GD2) that spontaneously clusters on the surface of human T cells in the absence of antigen and mimics chronic antigen exposure leading to T cell exhaustion. Using this model, we demonstrate that exhausted CD39+CD8+ CAR T cells actively produce adenosine and mediate immune suppression. In an attempt to generate adenosine resistance and enhance the function of exhausted CAR T cells, we knocked out CD39, CD73, or A2aR (adenosine A2a receptor) but observed only modest changes in phenotype and transcriptomics. In contrast, overexpression of transmembrane-bound adenosine deaminase, which metabolizes adenosine to inosine, induced higher frequency of stem- and central- like memory T cells, and a simultaneous decrease of exhausted T cell subpopulations. Direct exposure of HA-GD2 CAR T cells to high inosine concentration during cell manufacturing process, lead to a higher frequency of central-like memory cells and significant fitness enhancement associated with broad changes at the metabolic level. RNAseq and cyTOF analysis indicated decreased glycolytic flux, increased mitochondrial activity driven by glutamine and polyamine metabolism. Further, inosine altered the epigenetic state of HA-GD2 CAR T cells. We observed significant enrichment of IRF and NF-κB transcription factor motifs and motifs associated with memory differentiation in T cells grown in the presence of inosine. Finally, we showed that production of exhausted HA-GD2 and clinical GD2 CAR T cells in inosine-containing culture media enhances their in vivo efficacy, leading to improved survival in GD2+ tumor-bearing mice. In conclusion, we propose introducing inosine during GMP cell manufacturing as a novel strategy to improve CAR T function and subsequent clinical outcomes of CAR T cell therapy. Citation Format: Dorota Klysz, Katie Freitas, Meena Malipatlolla, Stefanie Maier, Carley Fowler, Louai Labanieh, Lucille Stuani, Bence Daniel, Katalin Sandor, Peng Xu, Jing Huang, Amaury Leruste, Vimal Keerthi, Nikolaos Gkitsas, Shabnum Patel, Justin Arredondo-Guerrero, Chris Fisher, Kyle Asano, Sunny Patel, Ansuman Satpathy, Kara Davis, Elena Sotillo, Steven Feldman, Crystal Mackall. Inosine endows CAR T cells with features of increased stemness and anti-tumor potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1158.

Abstract 1152: NKTR-255, a polymer-conjugated IL-15, dramatically improves ROR1 CAR-T cell persistence and anti-tumor efficacy in an autochthonous model of ROR1+ lung cancer

Abstract CAR-T cells have mediated remarkable clinical responses against hematological malignancies, but this success has yet to extend to solid tumors, where many factors in the tumor microenvironment (TME) inhibit their persistence and function. In a phase 1 trial at our Center, CAR-T cells targeting the tumor-associated antigen ROR1 in patients with non-small cell lung cancer (NSCLC) and triple negative breast cancer rapidly became dysfunctional and persisted poorly at tumor sites, ultimately failing to mediate objective responses. To evaluate strategies to improve ROR1 CAR-T efficacy, we adapted the KrasLSL-G12D/+;p53fl/fl (KP) autochthonous model of lung adenocarcinoma to express ROR1 (KPROR1). This aggressive model mimics the initiation, progression, and suppressive TME of human NSCLC. ROR1 CAR-T cells in KPROR1 mice rapidly declined in number, lost the ability to produce pro-inflammatory cytokines, and failed to significantly enhance tumor control, recapitulating the major barriers to CAR-T efficacy we observed in patients. NKTR-255 is a novel immunotherapeutic consisting of a polymer conjugated recombinant human interleukin-15 (rhIL-15) that could enhance the activity of CAR-T cells. NKTR-255 binds to the IL-15 receptor and IL-2/IL-15 receptor, thus maintaining the full potential spectrum of IL-15 biology while exerting a sustained effect on CD8+ memory T cell and NK cell expansion and persistence. We hypothesized that the combination of CAR-T cells and NKTR-255 would be feasible and safe and may extend the duration of efficacy of ROR1 CAR-T cells in ROR1+ lung tumors by improving their expansion and persistence. To test this, we treated tumor-bearing KPROR1 mice with control or ROR1 CAR-T cells with or without weekly injections of NKTR-255. NKTR-255 treatment resulted in a robust expansion of ROR1 CAR-T cells in the peripheral blood relative to vehicle-treated mice, which correlated with increased CAR-T cell infiltration into lung tumors. CAR-T cells in NKTR-255-treated mice also showed reduced signs of T cell exhaustion, including reduced expression of inhibitory receptors and enhanced ability to produce effector cytokines upon ex vivo re-stimulation. Importantly, combination therapy with both NKTR-255 and ROR1 CAR-T cells resulted in significantly improved tumor control relative to either treatment alone. Together, our work shows that NKTR-255 dramatically improves the persistence, function, and anti-tumor activity of ROR1 CAR-T cells in an aggressive autochthonous model of ROR1+ lung cancer. Our findings provide rationale to combine NKTR-255 with CAR-T cell therapy as a strategy to enhance the anti-tumor efficacy of CAR-T cells in patients with solid tumors. Citation Format: W. Sam Nutt, Takahiro Miyazaki, Mario Marcondes, Stanley R. Riddell, Shivani Srivastava. NKTR-255, a polymer-conjugated IL-15, dramatically improves ROR1 CAR-T cell persistence and anti-tumor efficacy in an autochthonous model of ROR1+ lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1152.

Transforming Cold Tumors into Hot Ones with a Metal-Organic Framework-Based Biomimetic Nanosystem for Enhanced Immunotherapy.

Immunotherapy has revolutionized the landscape in clinical tumor therapy, although the response rates in "cold" tumors are relatively low owing to the complex tumor microenvironment (TME). Cyclic guanosine monophosphate-adenosine monophosphate synthase/stimulator of interferon genes (cGAS/STING) pathway-inducing agents can reprogram the TME; however, their applications remain underutilized. Herein, we engineered a facile manganese-based metal-organic framework (Mn-MOF) encapsulating polyphyllin I (PPI) and coated it with red blood cell (RBC) membranes (RBC@Mn-MOF/PPI) that enhanced the cGAS/STING-mediated antitumor immunity. RBC@Mn-MOF/PPI was engineered by camouflaging it with a biomimetic RBC membrane for prolonged blood circulation and immune escape, which was also extended with TME-sensitive properties for triggering the release of PPI and Mn2+ to remodel the suppressive TME and augment antitumor immune responses. Furthermore, RBC@Mn-MOF/PPI helped transform cold tumors into "hot" ones by activating immune cells, as evidenced via dendritic cell maturation, cytotoxic T lymphocyte infiltration, and natural killer cell recruitment, thereby targeting primary and abscopal tumors and lung metastatic nodules. Therefore, our engineered nanosystem represents a novel strategy to transform immunologically "cold" tumors into "hot" ones by activating the cGAS/STING pathway, thereby addressing the major challenges associated with immunotherapy.

Z-Endoxifen prevents aggressive mammary cancers in mice by inhibiting cell proliferation and creating a tumor suppressive microenvironment.

Aggressive estrogen receptor (ER) positive breast cancer is frequently tamoxifen-resistant; alternative endocrine approaches exist for therapy, but not for prevention, particularly in premenopausal women. We examined the efficacy of the selective ER modulator (Z-endoxifen) as monotherapy and in combination with the selective progesterone receptor modulators (onapristone and ulipristal acetate) in the tamoxifen-insensitive C3(1)/SV40TAg mouse mammary tumorigenesis model. Unlike tamoxifen at human equivalent dose (HED) 101 mg/day, endoxifen at HED 24 mg/day significantly increased latency and reduced tumor growth relative to untreated controls. Ulipristal acetate (UPA) at HED 81 mg/day also significantly increased latency however failed to inhibit tumor growth, while onapristone (HED 98 mg/day) had no tumor prevention efficacy in this model. Addition of UPA to endoxifen did not enhance preventive efficacy over endoxifen alone. The expression of genes associated with cell cycle, cell proliferation and extracellular matrix remodeling was similarly repressed by endoxifen and UPA however only endoxifen significantly downregulated prominent genes associated with poor prognosis (Col11a1, Il17b, Pdgfa, Tnfrsf11a). Our results indicate that endoxifen can prevent breast cancers, even when tamoxifen-resistant, through its role in favorable tissue remodeling and immunomodulation.

Targeting myeloid-derived suppressor cells in tumor immunotherapy: Current, future and beyond.

Myeloid-derived suppressor cells (MDSCs) are one of the major negative regulators in tumor microenvironment (TME) due to their potent immunosuppressive capacity. MDSCs are the products of myeloid progenitor abnormal differentiation in bone marrow, which inhibits the immune response mediated by T cells, natural killer cells and dendritic cells; promotes the generation of regulatory T cells and tumor-associated macrophages; drives the immune escape; and finally leads to tumor progression and metastasis. In this review, we highlight key features of MDSCs biology in TME that are being explored as potential targets for tumor immunotherapy. We discuss the therapies and approaches that aim to reprogram TME from immunosuppressive to immunostimulatory circumstance, which prevents MDSC immunosuppression activity; promotes MDSC differentiation; and impacts MDSC recruitment and abundance in tumor site. We also summarize current advances in the identification of rational combinatorial strategies to improve clinical efficacy and outcomes of cancer patients, via deeply understanding and pursuing the mechanisms and characterization of MDSCs generation and suppression in TME.

Fusobacterium nucleatum induces a tumor microenvironment with diminished adaptive immunity against colorectal cancers.

Fusobacterium nucleatum (FN) plays a pivotal role in the development and progression of colorectal cancer by modulating antitumor immune responses. However, the impact of FN on immune regulation in the tumor microenvironment has not been fully elucidated. The abundance of FN was measured in 99 stage III CRC tumor tissues using quantitative polymerase chain reaction. Gene expression profiles were assessed and annotated using consensus molecular subtypes (CMS), Gene Ontology (GO) analysis, and deconvolution of individual immune cell types in the context of FN abundance. Immune profiling for tumor infiltrating T cells isolated from human tumor tissues was analyzed using flow cytometry. Ex vivo tumor-infiltrating T cells were stimulated in the presence or absence of FN to determine the direct effects of FN on immune cell phenotypes. Gene expression profiles, CMS composition, abundance of immune cell subtypes, and survival outcomes differed depending on FN infection. We found that FN infection was associated with poorer disease-free survival and overall survival in stage III CRC patients. FN infection was associated with T cell depletion and enrichment of exhausted CD8+ and FoxP3+ regulatory T cells in the tumor microenvironment. The presence of FN in tumors was correlated with a suppressive tumor microenvironment in a T cell-dependent manner. FN enhanced the suppressive immune microenvironment with high depletion of CD8+ T cells and enrichment of FoxP3+ regulatory T cells in human colorectal cancer cases. Our findings suggest a potential association for FN in adaptive immunity, with biological and prognostic implications.

TFAM deficiency in dendritic cells leads to mitochondrial dysfunction and enhanced antitumor immunity through cGAS-STING pathway

BackgroundMitochondrial transcription factor A (TFAM) is a transcription factor that maintains mitochondrial DNA (mtDNA) stabilization and initiates mtDNA replication. However, little is known about the immune regulation function and TFAM...

Nanoscale, antigen encounter-dependent, IL-12 delivery by CAR T cells plus PD-L1 blockade for cancer treatment

BackgroundChimeric antigen receptor (CAR)-T cell therapies for the treatment of hematological malignancies experienced tremendous progress in the last decade. However, essential limitations need to be addressed to further improve efficacy and reduce toxicity to assure CAR-T cell persistence, trafficking to the tumor site, resistance to an hostile tumor microenvironment (TME), and containment of toxicity restricting production of powerful but potentially toxic bioproducts to the TME; the last could be achieved through contextual release upon tumor antigen encounter of factors capable of converting an immune suppressive TME into one conducive to immune rejection.MethodsWe created an HER2-targeting CAR-T (RB-312) using a clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) system, which induces the expression of the IL-12 heterodimer via conditional transcription of its two endogenous subunits p35 and p40. This circuit includes two lentiviral constructs. The first one (HER2-TEV) expresses an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3z co-stimulatory domains linked to the tobacco etch virus (TEV) protease and two single guide RNAs (sgRNA) targeting the interleukin (IL)-12A and IL12B transcription start site (TSS), respectively. The second construct (LdCV) encodes linker for activation of T cells (LAT) fused to nuclease-deactivated Streptococcus Pyogenes Cas9 (dCas9)-VP64-p65-Rta (VPR) via a TEV-cleavable sequence (TCS). Activation of the CAR brings HER2-TEV in close proximity to LdCV releasing dCas9 for nuclear localization. This conditional circuit leads to conditional and reversible induction of the IL-12/p70 heterodimer. RB-312 was compared in vitro to controls (cRB-312), lacking the IL-12 sgRNAs and conventional HER2 CAR (convCAR).ResultsThe inducible CRISPRa system activated endogenous IL-12 expression resulting in enhanced secondary interferon (FN)-γ production, cytotoxicity, and CAR-T proliferation in vitro, prolonged in vivo persistence and greater suppression of HER2+ FaDu oropharyngeal cancer cell growth compared to the conventional CAR-T cell product. No systemic IL-12 was detected in the peripheral circulation. Moreover, the combination with programmed death ligand (PD-L1) blockade demonstrated robust synergistic effects.ConclusionsRB-312, the first clinically relevant product incorporating a CRISPRa system with non-gene editing and reversible upregulation of endogenous gene expression that promotes CAR-T cells persistence and effectiveness against HER2-expressing tumors. The autocrine effects of reversible, nanoscale IL-12 production limits the risk of off-tumor leakage and systemic toxicity.

Oncolytic BHV-1 Is Sufficient to Induce Immunogenic Cell Death and Synergizes with Low-Dose Chemotherapy to Dampen Immunosuppressive T Regulatory Cells.

Immunogenic cell death (ICD) can switch immunologically "cold" tumors "hot", making them sensitive to immune checkpoint inhibitor (ICI) therapy. Many therapeutic platforms combine multiple modalities such as oncolytic viruses (OVs) and low-dose chemotherapy to induce ICD and improve prognostic outcomes. We previously detailed many unique properties of oncolytic bovine herpesvirus type 1 (oBHV) that suggest widespread clinical utility. Here, we show for the first time, the ability of oBHV monotherapy to induce bona fide ICD and tumor-specific activation of circulating CD8+ T cells in a syngeneic murine model of melanoma. The addition of low-dose mitomycin C (MMC) was necessary to fully synergize with ICI through early recruitment of CD8+ T cells and reduced infiltration of highly suppressive PD-1+ Tregs. Cytokine and gene expression analyses within treated tumors suggest that the addition of MMC to oBHV therapy shifts the immune response from predominantly anti-viral, as evidenced by a high level of interferon-stimulated genes, to one that stimulates myeloid cells, antigen presentation and adaptive processes. Collectively, these data provide mechanistic insights into how oBHV-mediated therapy modalities overcome immune suppressive tumor microenvironments to enable the efficacy of ICI therapy.

Precision Delivery of Dual Immune Inhibitors Loaded Nanomodulator to Reverse Immune Suppression for Combinational Photothermal-Immunotherapy.

Although photothermal therapy (PTT) can noninvasively kill tumor cells and exert synergistic immunological effects, the immune responses are usually harmed due to the lack of cytotoxic T cells (CTLs) pre-infiltration and co-existing of intricate immunosuppressive tumor microenvironment (TME), including the programmed cell death ligand 1 (PD-L1)/cluster of differentiation 47 (CD47)/regulatory T cells (Tregs)/M2-macrophages overexpression. Indoleamine 2, 3-dioxygenase inhibitor (NLG919) or bromodomain extra-terminal inhibitor (OTX015) holds great promise to reprogram suppressive TME through different pathways, but their collaborative application remains a formidable challenge because of the poor water solubility and low tumor targeting. To address this challenge, a desirable nanomodulator based on dual immune inhibitors loaded mesoporous polydopamine nanoparticles is designed. This nanomodulator exhibits excellent biocompatibility and water solubility, PTT, and bimodal magnetic resonance/photoacoustic imaging abilities. Owing to enhanced permeability and retention effect and tumor acidic pH-responsiveness, both inhibitors are precisely delivered and locally released at tumor sites. Such a nanomodulator significantly reverses the immune suppression of PD-L1/CD47/Tregs, promotes the activation of CTLs, regulates M2-macrophages polarization, and further boosts combined therapeutic efficacy, inducing a strong immunological memory. Taken together, the nanomodulator provides a practical approach for combinational photothermal-immunotherapy, which may be further broadened to other "immune cold" tumors.

Emerging Trends in Nano-Driven Immunotherapy for Treatment of Cancer.

Despite advancements in the development of anticancer medications and therapies, cancer still has the greatest fatality rate due to a dismal prognosis. Traditional cancer therapies include chemotherapy, radiotherapy, and targeted therapy. The conventional treatments have a number of shortcomings, such as a lack of selectivity, non-specific cytotoxicity, suboptimal drug delivery to tumour locations, and multi-drug resistance, which results in a less potent/ineffective therapeutic outcome. Cancer immunotherapy is an emerging and promising strategy to elicit a pronounced immune response against cancer. Immunotherapy stimulates the immune system with cancer-specific antigens or immune checkpoint inhibitors to overcome the immune suppressive tumour microenvironment and kill the cancer cells. However, delivery of the antigen or immune checkpoint inhibitors and activation of the immune response need to circumvent the issues pertaining to short lifetimes and effect times, as well as adverse effects associated with off-targeting, suboptimal, or hyperactivation of the immune system. Additional challenges posed by the tumour suppressive microenvironment are less tumour immunogenicity and the inhibition of effector T cells. The evolution of nanotechnology in recent years has paved the way for improving treatment efficacy by facilitating site-specific and sustained delivery of the therapeutic moiety to elicit a robust immune response. The amenability of nanoparticles towards surface functionalization and tuneable physicochemical properties, size, shape, and surfaces charge have been successfully harnessed for immunotherapy, as well as combination therapy, against cancer. In this review, we have summarized the recent advancements made in choosing different nanomaterial combinations and their modifications made to enable their interaction with different molecular and cellular targets for efficient immunotherapy. This review also highlights recent trends in immunotherapy strategies to be used independently, as well as in combination, for the destruction of cancer cells, as well as prevent metastasis and recurrence.

Small-molecule toosendanin reverses macrophage-mediated immunosuppression to overcome glioblastoma resistance to immunotherapy.

T cell-based immunotherapy holds promise for treating solid tumors, but its therapeutic efficacy is limited by intratumoral immune suppression. This immune suppressive tumor microenvironment is largely driven by tumor-associated myeloid cells, including macrophages. Here, we report that toosendanin (TSN), a small-molecule compound, reprograms macrophages to enforce antitumor immunity in glioblastoma (GBM) in mouse models. Our functional screen of genetically probed macrophages with a chemical library identifies that TSN reverses macrophage-mediated tumor immunosuppression, leading to enhanced T cell infiltration, activation, and reduced exhaustion. Chemoproteomic and structural analyses revealed that TSN interacts with Hck and Lyn to abrogate suppressive macrophage immunity. In addition, a combination of immune checkpoint blockade and TSN therapy induced regression of syngeneic GBM tumors in mice. Furthermore, TSN treatment sensitized GBM to Egfrviii chimeric antigen receptor (CAR) T cell therapy. These findings suggest that TSN may serve as a therapeutic compound that blocks tumor immunosuppression and circumvents tumor resistance to T cell-based immunotherapy in GBM and other solid tumors that warrants further investigation.