LLC Tumor Research Articles

Abstract 2423: Image-guided interstitial photodynamic therapy with palliative radiotherapy for ablating extrabronchial malignant central airway obstruction

Abstract Background: Patients with extrabronchial tumors that induce malignant central airway obstruction (MCAO) face dire prognoses with significant morbidity and 1-7 months overall survival. These tumors are inoperable. High-dose curative radiotherapy is associated with significant toxicity. Chemotherapy has limited localized benefits and immunotherapy cannot address the immediate need to halt tumor progression. Our preliminary data suggest that image-guided interstitial photodynamic therapy (I-PDT) with porfimer sodium is safe and potentially effective in patients with extrabronchial tumors that induce MCAO and there is a need to further improve the rate of response. Here we present results showing an improved tumor response rate by augmenting I-PDT with palliative radiotherapy (p-XRT). Methods: We investigated the addition of p-XRT prior to I-PDT to sensitize the target tumor to subsequent photoreaction in mouse models. Eight- to twelve-week-old C3H and C57B/6 mice with locally advanced (400-600 mm3) SCC-VII or LLC tumors, respectively, were treated with I-PDT alone or with p-XRT followed by I-PDT. The p-XRT regimen was 10 Gy x2 over two days. At 24-26 h after the second 10 Gy fraction, the I-PDT was administered with 5 mg/kg porfimer sodium activated with 630-nm laser light at 5.0 mW/cm2 and 45 J/cm2 at tumor margins. Photoacoustic imaging and light dosimetry measurements were used to investigate the impact of p-XRT on the tumor microenvironment as it pertains to the response to I-PDT. Tumor progression was monitored over 60 days. Image-based treatment planning was used to translate the preclinical findings into a treatment in clinical settings (8 Gy x1 followed by I-PDT) for patients with extrabronchial MCAO. Results: Preceding I-PDT with p-XRT increases tumor oxygenation for 1-2 days after the last radiation fraction and improves light penetration within mouse tumors. Administering p-XRT 24-48 h before I-PDT significantly (p<0.05, log-rank test) increases the rate of complete tumor response and control from 0% in p-XRT alone or 0-6% in I-PDT alone, to 31-64% for p-XRT followed by I-PDT. In patients, administration of I-PDT with porfimer sodium 48 h after 8Gy x1 p-XRT is feasible and can provide control of extrabronchial MCAO accompanied by symptomatic relief. Conclusions: Augmenting I-PDT with p-XRT improves the rate of tumor response. Murine studies show that p-XRT increases tumor blood oxygenation and light penetration which could contribute to better response to I-PDT. I-PDT with porfimer sodium preceded by p-XRT is feasible with possible benefits in patients with extrabronchial MCAO. Citation Format: Emily Oakley, Sarah Chamberlain, Nathaniel Ivanick, Alan Hutson, Theresa Busch, Gal Shafirstein. Image-guided interstitial photodynamic therapy with palliative radiotherapy for ablating extrabronchial malignant central airway obstruction [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 2423.

Abstract 5108: Overcoming roadblocks of immunotherapy in non-small cell lung cancer

Abstract Non-small cell lung cancer (NSCLC) is one of the major causes of cancer-related death worldwide. Immunotherapy has helped to increase the survival of NSCLC patients, but the therapeutic response is limited to 23-26% of patients. While many factors impact immunotherapy failure, we have focused on a few, (a) heterogeneous expression of immune checkpoints (ICs) limiting the use of single immune checkpoint inhibitor (ICI) for everyone, (b) additional ICs responsible for blocking innate immune system and lack of antigen presentation to the T cells, and (c) lack of rationally designed combination therapies.Commonly used immunotherapies for NSCLC block PD1-PDL1 interaction, but the majority of the NSCLC patient does not have high PDL1 expression indicating PD1-PDL1 as a subsidiary mode of immune evasion. So, it is necessary to include additional ICIs in order to achieve adequate objective response in all NSCLC patients. In order to investigate the immune checkpoint expression, we screened several NSCLC patient-derived tumor tissues, and only 33% of them showed high expression of PDL1, whereas 64.6% of patients had high CD47 expression. CD47, an innate immune regulator, is overexpressed in lung cancer cells and delivers an “eat-me-not” signal by binding to signal regulatory protein alpha (SIRPα) in macrophage resulting in diminishing phagocytosis and antigen presentation to the T cells.Combining anticancer drugs with immunotherapy agents has become a popular approach to achieve a less toxic and long-acting objective therapeutic response. Here, we introduce antibody conjugated drug loaded nanotherapeutics (ADNs) consisting of a targeted therapy drug, phosphatidylinositol 3-kinase (PI3K) inhibitor PI103, and decorated with two ICIs for CD47 and PDL1. The ADN particles have been designed based on classical liposomal supramolecular nanoparticles followed by surface functionalization by the specific antibody. The anti-CD47-PDL1-ADN has shown increased cellular internalization and delivery of PI103 than IgG-ADN and anti-PDL1-ADN. The anti-CD47-PDL1-ADN showed low hematotoxicity and higher cell-killing potency for the cancer cells. The bispecific anti-CD47-PDL1-ADNs can block PD1-PDL1 and SIRPα-CD47 immune checkpoint interaction, activating both the innate (macrophage) and adaptive (T cells) immune responses. Reduced tumor growth and higher survival probability have been observed in the syngeneic LLC tumor model for anti-CD47-PDL1-ADN than monotherapy and traditional immunotherapy.In summary, we have introduced a lung cancer treatment strategy that can be an effective therapy for NSCLC patients, irrespective of the PDL1 expression level. The strategy combining bispecific immunotherapy and targeted therapy for activating the innate and adaptive immune systems and delivering targeted therapy drugs can emerge as a significant advance in the treatment of NSCLC patients. Citation Format: Tanmoy Saha, Shiladitya Sengupta. Overcoming roadblocks of immunotherapy in non-small cell 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 5108.

Abstract 1546: ADCC enhanced anti-CD25 mAb (TST010) demonstrated antitumor activity via depleting Treg cells and increasing CD8+T/Treg ratio in preclinical tumor models

Abstract Regulatory T (Treg) cells are a distinctive lineage of CD4+ T cells as characterized by expression of FoxP3 and CD25 markers. Treg cells are involved in tumor development and progression by inhibiting antitumor immunity. Accumulation of Treg cells in the tumor microenvironment (TME) is often associated with an adverse prognosis in multiple cancers and also has implication for predicting response to immunotherapy. Treg cell depletion strategy has been explored in preclinical and clinical settings to induce effective antitumor immune responses in TME. CD25, a high-affinity binding subunit alpha of the IL-2 receptor, is constitutively and highly expressed on Treg cells but also transiently upregulated on effector T cells. The high level of CD25 expression on Treg cells could deprive of IL-2 for effector T cells and inhibit their proliferation. Thus, an effective approach to selectively suppress Treg cell function is to deplete Treg cells while avoid blocking IL-2 binding to CD25 for allowing T effect cell proliferation and activation. Using our immune tolerance breaking antibody platform, we generated a humanized IgG1 subtype anti-CD25 monoclonal antibody (TST010). TST010 bound to human CD25 with high affinity and selectivity but did not block IL-2 binding to CD25. No blocking activity to IL-2 signaling on human PBMC was further confirmed by the phosphorylation of Stat5 using FACS analysis. By reducing fucosylation during cell culture process, TST010 mAb gained the enhanced ADCC activity. In an ADCC reporter assay, TST010 showed its sub-nanomolar ADCC activity against CD25-overexpression SU-DHL-1 cell line and human induced Treg cells, but much less to human activated CD8+ T effect cells due to lower CD25 expression. Similarly, in a NK cytotoxicity assay, TST010 selectively killed human induced Treg cells in contrast to activated CD8+ T cells. In vivo antitumor activity of TST010 was evaluated in the syngeneic mouse tumor models. The mouse colon cancer MC38 model was developed on the human CD25 knock-in mice. In the MC38 tumor model, TST010 markedly reduced the Treg (FoxP3+CD4+) population in both peripheral blood and TME. Importantly, the CD8+T/Treg ratio in tumor was increased starting at 2 weeks post TST010 treatment, and it resulted in a significant single agent tumor growth inhibition at a 10 mg/kg dose. Furthermore, in the LLC tumor model which does not respond well to checkpoint inhibitors, the anti-CD25 mAb showed a good combination effect with a PD-L1 inhibitor. In summary, we have discovered a novel therapeutic anti-CD25 mAb that can deplete Treg cells without blocking IL-2 signaling. As demonstrated in our preclinical tumor models, it has a good potential to induce effective antitumor immune responses in TME and tumor growth inhibition especially in combination with PD-1/PD-L1 treatment. Citation Format: Di Sun, Huanhuan Guo, Hongjun Li, Xinlai Yao, Yiqing Xu, Shenjie Zhang, Shijie Zhou, Xiaoli Zi, Lisa Zheng, Yi Gu, Xueming Qian. ADCC enhanced anti-CD25 mAb (TST010) demonstrated antitumor activity via depleting Treg cells and increasing CD8+T/Treg ratio in preclinical tumor models [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 1546.

The Voltage-Gated Hv1 H+ Channel Is Expressed in Tumor-Infiltrating Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSCs) are key determinants of the immunosuppressive microenvironment in tumors. As ion channels play key roles in the physiology/pathophysiology of immune cells, we aimed at studying the ion channel repertoire in tumor-derived polymorphonuclear (PMN-MDSC) and monocytic (Mo-MDSC) MDSCs. Subcutaneous tumors in mice were induced by the Lewis lung carcinoma cell line (LLC). The presence of PMN-MDSC (CD11b+/Ly6G+) and Mo-MDSCs (CD11b+/Ly6C+) in the tumor tissue was confirmed using immunofluorescence microscopy and cells were identified as CD11b+/Ly6G+ PMN-MDSCs and CD11b+/Ly6C+/F4/80-/MHCII- Mo-MDSCs using flow cytometry and sorting. The majority of the myeloid cells infiltrating the LLC tumors were PMN-MDSC (~60%) as compared to ~10% being Mo-MDSCs. We showed that PMN- and Mo-MDSCs express the Hv1 H+ channel both at the mRNA and at the protein level and that the biophysical and pharmacological properties of the whole-cell currents recapitulate the hallmarks of Hv1 currents: ~40 mV shift in the activation threshold of the current per unit change in the extracellular pH, high H+ selectivity, and sensitivity to the Hv1 inhibitor ClGBI. As MDSCs exert immunosuppression mainly by producing reactive oxygen species which is coupled to Hv1-mediated H+ currents, Hv1 might be an attractive target for inhibition of MDSCs in tumors.

RIG-I-mediated innate immune signaling in tumors reduces the therapeutic effect of oncolytic vesicular stomatitis virus.

Oncolytic viral therapy is a promising method for tumor treatment. Currently, several oncolytic viruses (OVs) have been used as tumor therapy at different phases of research and clinical trials. OVs not only directly lyse tumor cells due to viral replication but also initiate host antitumor immune responses. Previous studies have primarily focused on how OVs activate adaptive immune responses in immune cells. However, the role of innate immune responses in tumors induced by OVs remains unclear. To determine the innate immune responses induced by vesicular stomatitis virus (VSV), the mutant VSVΔM51 strain was used for the infection and quantitative polymerase chain reaction (qPCR) was employed to measure the transcriptional levels of antiviral genes. The knockdown efficiency of RIG-I was examined by qPCR. Viral titers were measured by plaque assays. Tumor models were established by intradermally implanting RIG-I-knockdown and control LLC cells into the flank of wild type C57BL/6J mice. When the tumors reached approximately 50mm3 , they were infected with VSVΔM51 via intratumoral injections to examine its therapeutic effect. Infection with VSVΔM51 triggered remarkable innate immune responses in several tumor cell lines through the cytoplasmic RIG-I sensing pathway. Moreover, we found that intratumoral injection of VSVΔM51 effectively reduced tumor growth in murine LCC lung cancer model. Importantly, VSVΔM51 -induced antitumor therapy was more effective in murine LLC tumor model established using Rig-I-knockdown cells compared with the tumor model established using control cells. RIG-I-mediated innate immune signaling in tumor cells plays a negative role in regulating antitumor therapy with VSVΔM51 virus.

Conjugation of Listeriolysin O to Anti-CD47 mAb: Harnessing Listeria Toxin to Weaponize Macrophages against Tumor Cells

<h3>Purpose/Objective(s)</h3> CD47 is expressed on various tumor types including breast, prostate, glioblastoma, non-Hodgkin lymphoma, and hepatocellular carcinoma. Interrupting the CD47-SIRP alpha axis by monoclonal antibodies fosters macrophage-mediated phagocytosis of solid and hematopoietic tumor cells. The pore-forming toxin listeriolysin O (LLO) is activated in the acidic vacuoles of bone marrow-derived macrophages. We hypothesize that LLO conjugation to the anti-CD47 mAb enhances the release of autophagic cargo within macrophages thereby augmenting cGAS-STING activation, antigen presentation, and tumor phagocytosis. <h3>Materials/Methods</h3> The anti-CD47 mAb was conjugated to the LLO toxin using a water-soluble SPDP crosslinker and purified by affinity chromatography. The cytotoxicity of anti-CD47-LLO mAb relative to anti-CD47 mAb was tested in the mouse breast cancer cell line E0771, the human breast cancer cell line MDA-MB-468, the mouse lung cancer cell line LLC, and the human leukemia monocytic cell line THP-1. C57B6 mouse bone marrow-derived macrophages (BMDM) co-cultured with tumor cell lines were analyzed in the presence of anti-CD47-LLO mAb and anti-CD47 mAb to determine the efficiency of BMDM tumor cell phagocytosis, STING activation, and antigen presentation. Tumor growth of E0771-bearing wildtype mice was measured following intratumoral injection of anti-CD47 mAb and anti-CD47-LLO mAB. <h3>Results</h3> The anti-CD47-LLO mAb conjugate enhanced BMDM phagocytosis of LLC and E0771 tumor cells in a dose dependent manner that was significantly higher than cells treated with the anti-CD47 mAb alone. The anti-CD47-LLO mAb significantly increased BMDM levels of phosphorylated STING, interferon-stimulated genes, and antigen presentation relative to anti-CD47 mAb treatment. At doses compatible with preserved animal total body weight, red blood cell counts, and lymphocyte counts, intratumoral injection of anti-CD47-LLO mAb significantly suppressed tumor growth of E0771-bearing wildtype mice relative to the anti-CD47 mAb. <h3>Conclusion</h3> Conjugation of the LLO toxin to anti-CD47 mAb enhances macrophage cGAS-STING activation, antigen presentation, and tumor cell phagocytosis. This novel CD47 antibody builds on clinical interest in targeting macrophages for the treatment of malignancy and may be studied as a supplemental therapy for patients with tumors resistant or refractory to checkpoint therapy.

A Novel Therapeutic Tumor Vaccine Targeting MUC1 in Combination with PD-L1 Elicits Specific Anti-Tumor Immunity in Mice.

Dendritic cells (DCs), as professional antigen-presenting cells (APCs), play a key role in the initiation and regulation of humoral and cellular immunity. DC vaccines loaded with different tumor-associated antigens (TAAs) have been widely used to study their therapeutic effects on cancer. A number of clinical trials have shown that DCs are safe as an antitumor vaccine and can activate certain anti-tumor immune responses; however, the overall clinical efficacy of DC vaccine is not satisfactory, so its efficacy needs to be enhanced. MUC1 is a TAA with great potential, and the immune checkpoint PD-L1 also has great potential for tumor treatment. Both of them are highly expressed on the surface of various tumors. In this study, we generated a novel therapeutic MUC1-Vax tumor vaccine based on the method of PD-L1-Vax vaccine we recently developed; this novel PD-L1-containing MUC1-Vax vaccine demonstrated an elevated persistent anti-PD-L1 antibody production and elicited a much stronger protective cytotoxic T lymphocyte (CTL) response in immunized mice. Furthermore, the MUC1-Vax vaccine exhibited a significant therapeutic anti-tumor effect, which significantly inhibited tumor growth by expressing a high MUC1+ and PD-L1+ level of LLC and Panc02 tumor cells, and prolonged the survival of cancer-bearing animals. Taken together, our study provides a new immunotherapy strategy for improving the cross-presentation ability of therapeutic vaccine, which may be applicable to pancreatic cancer, lung cancer and for targeting other types of solid tumors that highly express MUC1 and PD-L1.

Liposomal Co-delivery of PD-L1 siRNA/Anemoside B4 for Enhanced Combinational Immunotherapeutic Effect.

Combination therapy has gained a lot of attention thanks to its superior activity against cancer. In the present study, we report a cRGD-targeted liposomal preparation for co-delivery of programmed cell death ligand 1 (PD-L1) small interfering RNA (siRNA) and anemoside B4 (AB4)─AB4/siP-c-L─and evaluate its anticancer efficiency in mouse models of LLC and 4T1 tumors. AB4/siP-c-L showed a particle size of (180.7 ± 7.3) nm and a ζ-potential of (32.8 ± 1.5) mV, with high drug encapsulation, pH-sensitive release properties, and good stability in serum. AB4/siP-c-L demonstrated prolonged blood circulation and increased tumor accumulation. Elevated cellular uptake was dependent on the targeting ligand cRGD. This combination induced significant tumor inhibition in LLC xenograft tumor-bearing mice by downregulating PD-L1 protein expression and modulating the immunosuppressive microenvironment. Liposomes favored the antitumor T-cell response with long-term memory, without obvious toxicity. A similar tumor growth inhibition was also demonstrated in the 4T1 tumor model. In summary, our results indicate that cRGD-modified and AB4- and PD-L1 siRNA-coloaded liposomes have potential as an antitumor preparation, and this approach may lay a foundation for the development of a new targeted drug delivery system.

Abstract 218: Novel SOD mimetics GC4419 &amp; GC4711 induce synergistic tumoricidal effects combined with radiotherapy in lung cancer models

Abstract Introduction: Superoxide dismutase mimetics (SODm) synergize with radiation (IR) by converting radiolytically produced superoxide to hydrogen peroxide. Cancer cells lacking catalase function fail to clear H2O2 resulting in increased tumoricidal load. In addition, complex interactions through redox signaling and immune interplay are suggested mechanisms of action. Our pre-clinical studies with the SODm GC4419 (AVA) indicated that increased inflammatory, TNFα, and apoptosis signaling contribute to treatment synergy that was most effective at hypofractionation (Sci Transl Med. 13:593). The analogue GC4711 (GC) is currently in clinical trials for SBRT against NSCLC and pancreatic cancer. However, optimal combination schedules and molecular mechanisms are largely unknown. Here, we present optimized combination schedules using human lung tumor xenografts regarding drug administration timing and dosage, as well as local and abscopal tumor control in a syngeneic lung tumor model. Further, we tested various cell stress and immune pathways to identify targets of synergistic IR-drug interaction. Methods: Female athymic nude and C57BL/6 mice were inoculated with H1299 and LLC, respectively. H1299 tumors were irradiated with 10-15 Gy and GC was given once or twice daily (24 or 2x20 mg/kg, respectively) over 5 days, including untreated controls. LLC tumors were set up as an abscopal model and mice received combination treatment with GC and 15 Gy, 15 Gy only, or GC only, including untreated controls. In vitro, lung cancer cell lines were pre-treated with AVA or GC 30 min prior to IR and assayed for various immune or DNA damage-related endpoints. Results: The anti-tumor efficacy of single or double GC administration showed nonlinear dose-dependence indicating complex drug-IR interplay. Optimal drug administration appears to be around 3 hours prior to irradiation while time-of-day of treatment has no observable effect in this model. In the LLC double tumor model, GC in combination with IR induced significant and pronounced abscopal tumor control. In vitro, analysis revealed GC action related to e.g. 53BP1, yH2AX, Akt, R-loop formation and replication stress, and interaction with various immune pathways. Discussion and Conclusions: The radiotherapeutic effect can be vastly increased with superoxide dismutase mimetics, both within and outside the treatment field. Combination therapy requires screening of schedules regarding timing, drug dosage, and IR dose and fractionation to achieve optimal tumoricidal effects. SODm synergize with IR acting on various molecular targets that can be harnessed to overcome treatment resistance. Secondary tumor control is a promising new venue for IR-SODm combination therapy. Moreover, redox-immune interaction opens new therapeutic applications with combined immune checkpoint inhibitors for unprecedented tumor control and cure rate. Citation Format: Britta Langen, Laurentiu Pop, Zengfu Shang, Moito Iijima, James Nicholson, Mingming Yang, Michael D. Story. Novel SOD mimetics GC4419 &amp; GC4711 induce synergistic tumoricidal effects combined with radiotherapy in lung cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 218.

Abstract 4175: Combining a novel MEK inhibitor with immunomodulation to promote an anti-tumor response

Abstract To enhance the therapeutic scope of MEK inhibitors (MEKis), we aim to develop new strategies to extend their usage to MEKi resistant RAS mutant cancers, which represent an unmet clinical need. CH5126766 (CKI27) binds the allosteric site of MEK to inhibit its kinase activity but is novel due to its interaction with MEK S218 and 228, which blocks their phosphorylation by RAF. CKI27 bound MEK binds to RAF and cannot be released by phosphorylation, thus becoming a dominant negative inhibitor of RAF activation. This prevents the induction of MEK phosphorylation observed with other MEKis. Although this results in more potent tumor control, CKI27 is also capable of inhibiting T cell function because the MAPK/ERK pathway is activated downstream of T cell receptor signaling. We aim to balance the positive and negative immunomodulatory effects of MEKis for optimal combination with immunotherapy. We observed that CKI27 increased MHC expression on tumor cells and improved T cell mediated killing. Yet, CKI27 also decreased T cell proliferation, activation, and cytolytic activity. Intermittent administration of CKI27 allowed T cells to recover and partially relieved these inhibitory effects. Further combination with agonist antibodies anti-OX40 and GITR completely alleviated T cell inhibition and increased combination efficacy with immune checkpoint blockade antibody anti-CTLA-4. We also observed an increase in proliferation and T cell activation markers in LLC tumor bearing mice treated with the combination of CKI27, anti-GITR, and anti-CTLA-4. Understanding the immunomodulatory effects of combining CKI27 with immunotherapy will elucidate the mechanism behind this increased efficacy. This will allow us to make more informed decisions in dosing regimens, overcoming resistance, and generating long-term immune responses in future clinical trials treating patients with RAS mutant cancers. Citation Format: Lauren Dong, Hyejin Choi, Sadna Budhu, Isabell Schulze, Svena Verma, Nezar Mehanna, Neal Rosen, Taha Merghoub, Jedd Wolchok. Combining a novel MEK inhibitor with immunomodulation to promote an anti-tumor response [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 4175.

Abstract 3180: Targeting LRG1 to normalize tumor vasculature and enhance therapeutic efficacy

Abstract In recent years improving tumor vascular function, to render the tumour microenvironment less permissive and to improve delivery of therapeutics, has gained traction due to a growing body of supportive evidence. Identifying suitable targets that are tractable, ubiquitous and safe, however, has proven to be more challenging. Almost a decade ago we reported that a secreted glycoprotein, leucine-rich alpha-2-glycoprotein 1 (LRG1), was induced in ocular neovascular complications and contributed to the formation of dysfunctional neovessels1. More recently, we have reported that LRG1 is expressed in experimental and human tumors, and that its inhibition with a function-blocking antibody improves outcome in multiple primary2 and metastatic3 models of cancer. Crucially, we found that LRG1 blockade normalizes tumor vessels and enhances the efficacy of cisplatin, adoptive T cell and checkpoint inhibitor therapy, and that a humanized version of our blocking antibody named Magacizumab effectively inhibits tumour growth both alone and as an antibody-drug-conjugate4.Using animal models and in vitro assays we present here further data in support of LRG1 as a promising target for the treatment of solid cancers. We have further investigated the effects of LRG1 blockade on immune cell infiltration using flow cytometric and immunohistochemical analysis. In subcutaneous B16 melanoma-bearing mice treated with a PD1 checkpoint inhibitor, antibody blockade of LRG1 significantly enhanced the infiltration of CD3+ and CD8+ T cells, with the latter exhibiting a more activated phenotype as evidenced by higher GrzB, reduced PD1hi expression and increased proliferation. We also observed a reduction in the Tregs:Th ratio and a higher number of MHCII+ cells. These outcomes were also observed in LLC tumors alongside a reduction in the number of infiltrated neutrophils. In preliminary studies, where we investigated LRG1 blockade in the Rag1 mouse, we observed no effect suggesting that the downstream mode of action is mediated by impacting the immune system. Finally, to test the development of our humanized anti-LRG1 antibody Magacizumab, that only recognizes human LRG1, we demonstrated its efficacy in B16F0 tumors grown in a human LRG1 knock-in mouse. These studies provide compelling evidence that LRG1 is a novel, legitimate and potentially efficacious target for the treatment of various human solid cancers.

Abstract 245: Interleukin-4 receptor-targeted Abraxane inhibits tumor growth by enhancing drug delivery and reprogramming of M2-type macrophages into M1 phenotype

Abstract Abraxane, an albumin-bound paclitaxel nanoparticle, is widely used in treating patients with cancer such as breast cancer. The present study aims at a targeted delivery of Abraxane to tumors by conjugating it with IL4RPep-1, an interleukin-4 receptor (IL4R)-binding peptide. IL4R is up-regulated in many tumor cells and M2-polarized macrophages. IL4R-targeted Abraxane (IL4RPep-1-Abx) showed more efficient binding and internalization into IL4R-high tumor cells and M2-type macrophages than untargeted Abraxane, which was inhibited in part by an inhibitor of macropinocytotsis and receptor-mediated endocytosis, respectively, while being completely inhibited by an IL4R-blocking antibody. In the same context, IL4RPep-1-Abx induced higher levels of cytotoxicity to IL4R-high tumor cells and reprogramming of M2-type macrophages into M1-like phenotype than untargeted Abraxane. In vivo fluorescence imaging showed that IL4RPep-1-Abx homed to tumor, with little accumulation in the liver and lung, more efficiently than untargeted Abraxane. In addition, IL4RPep-1-Abx inhibited tumor growth and metastasis to the lung in 4T1 or LLC tumor-bearing mice and reduced the number of tumor nodules in the lung of K-ras transgenic mice more efficiently than untargeted Abraxane. Liver function was maintained within the normal ranges after treatment with IL4RPep-1-Abx. In 4T1 and LLC tumor tissues, IL4RPep-1-Abx reduced the population of immune-suppressive cells such as M2-type macrophages and increased that of immune-stimulatory cells such as CD8+ T cells compared with untargeted Abraxane. These results suggest that IL4R-targeted Abraxane can enhance the therapeutic efficacy and anti-tumor immunity of Abraxane and is a promising strategy for targeted cancer therapy. Citation Format: Poongkavithai Vadevoo Sri Murugan, Gunassekaran Gowri Rangaswamy, Byungheon Lee. Interleukin-4 receptor-targeted Abraxane inhibits tumor growth by enhancing drug delivery and reprogramming of M2-type macrophages into M1 phenotype [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 245.

Abstract 1307: Modulation of the immune environment in a preclinical orthotopic model of non-small cell lung cancer exposed to ablative doses of radiation

Abstract Stereotactic body radiation therapy (SBRT) allows the administration of ablative doses and is used for the management of a proportion of non-small cell lung cancer (NSCLC) patients. SBRT allows high fractional doses (≥ 8 Gy), which may result in a fundamentally different biological effect from conventional radiotherapy (RT). The effects of SBRT are mediated by cytotoxic, inflammatory and immunomodulatory mechanisms. The latter are of particular interest and their understanding is the key to potentiating the local effect and synergy with immunotherapies. Several studies have shown the involvement of the immune system in the antitumor efficacy of RT, but these studies were mainly conducted in subcutaneous murine tumor models using conventional doses of RT. In contrast, the effects of ablative irradiation (IR) doses on the infiltration and activity of immune cells are poorly understood, especially in orthotopic models.Here we used a murine orthotopic model of NSCLC to evaluate the effects of high doses of RT, delivered using a small animal radiation platform (SARRP) that allows stereotactic IR with a millimetric precision. Mice were subjected to a trans-pleural injection with Lewis lung carcinoma cells expressing luciferase (LLC-Luc) and irradiated at doses ranging from 8Gy to 60Gy in 1-3 fractions. Our results show that orthotopic LLC tumors, which correctly develop as single tumor mass within the lung parenchyma, are resistant to SBRT, with no complete tumor regression observed even after 60Gy IR. The characterization of the tumor environment by at the clinically relevant dose of 20Gy showed transient (by day 3 post-IR) increased CD8+ T cells and inflammatory monocytes within the tumor bed but not in the juxtatumor tissue. Resident alveolar macrophages where mostly detected in the juxtatumor tissue and were depleted by RT. Although RT did not affect interstitial tumor-associated macrophages (TAM) number, we observed that the fraction of CD206+ TAM was decreased together with an increased proportion of MHC class II+ TAM. At later time points, Treg harboring increased CD69 accumulated in the tumor bed and TAM recovered a similar phenotype compared to non-irradiated mice. The analysis of the tumor cytokinome revealed the modulation of several cytokines and chemokines including CCL-5, CXCL-10, LIF (Leukemia Inhibitory Factor) and Eotaxin. Altogether, these results argue that high IR doses induce transient and localized activation of the tumor immune environment that is rapidly dampened by anti-inflammatory cell recovery, such as Tregs and CD206 TAMs. The ongoing analyses using a single-cell RNAseq approach on sorted CD45+ cells will allow a precise evaluation of the composition and phenotype of the tumor immune populations.The results of this project will guide the design of therapeutic combinations with immunomodulatory agents to optimize the tumor response to SBRT. Citation Format: Céline Clémenson, Morgane Dos Santos, Maxime Petit, Jeremy Lavigne, Winchygn Liu, Marina Milic, Georges Tarlet, Agnès François, Alexandre Boissonnas, Fabien Milliat, Michele Mondini, Eric Deutsch. Modulation of the immune environment in a preclinical orthotopic model of non-small cell lung cancer exposed to ablative doses of radiation [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 1307.

Cancer cell-autonomous overactivation of PARP1 compromises immunosurveillance in non-small cell lung cancer

BackgroundHigh activity of poly(ADP-ribose) polymerase-1 (PARP1) in non-small cell lung cancer (NSCLC) cells leads to an increase in immunohistochemically detectable PAR, correlating with poor prognosis in patients with NSCLC, as...

Delivery of CXCL9/10/11 plasmid DNAs promotes the tumor-infiltration of T cells and synergizes with PD1 antibody for treating lung cancer

BackgroundImmune checkpoint blockade (ICB)-based cancer immunotherapy presents promising efficacy in cancer treatment. However, only a small portion of patients show responsiveness to the treatment, which is partially caused by limited tumor infiltration of T cells. Chemokines CXCL9, CXCL10 and CXCL11 bind to their receptor CXCR3 to regulate T cell invasion.MethodsWe delivered plasmids encoding CXCL9, CXCL10 and CXCL11 to tumor cells and tumor tissues using nanoparticles and investigated their effect on T cell invasion and infiltration. In addition, we applied these nanoparticles together with anti-PD-1 antibody, which is known to activate T cells and restore immune function against tumor cells. The anti-tumor effects were evaluated.ResultsDelivering plasmids encoding CXCL9, CXCL10 and CXCL11 by nanoparticles resulted in expression of these chemokines in both LLC cells and tumors. Expressing CXCL9, CXCL10 and CXCL11 promoted the infiltration of T cells in vitro and in vivo, as well as decreased the tumor size. Nanoparticles together with anti-PD-1 displayed the best anti-tumor effects.ConclusionsDelivery of CXCL9/10/11 plasmids by nanoparticles promoted T cell infiltration in tumors and synergizes with the activity of anti-PD1 antibody.

Functional antigen processing and presentation mechanism as a prerequisite factor of response to treatment with dendritic cell vaccines and anti-PD-1 in preclinical murine LLC1 and GL261 tumor models.

Low efficacy of cancer immunotherapy encourages the search for possible resistance mechanisms and biomarkers that would predict the outcome of immunotherapy in oncology patients. Most cancer immunotherapies act on T lymphocytes, which can specifically recognize and kill tumor cells. However, for immunotherapy-activated T lymphocytes to be able to perform these functions, proper tumor Ag processing and surface presentation by MHC-I molecule is important. Knowing the significance of Ag processing and presentation mechanism (APM) in anti-tumor immune response, we sought to evaluate how the functionality of APM affects tumor immune microenvironment and response to dendritic cell vaccines (DCV) and anti-PD-1. By comparing murine Lewis lung carcinoma LLC1 and glioma GL261 models a decreased expression of APM-related genes, such as Psmb8, Psmb9, Psmb10, Tap1, Tap2, Erap1, B2m, and low expression of surface MHC-I molecule were found in LLC1 cells. Changes in APM-related gene expression affected the ability of T lymphocytes to recognize and kill LLC1 cells, resulting in the absence of cytotoxic immune response and resistance to DCV and anti-PD-1. An emerging cytotoxic immune reaction and sensitivity to DCV and anti-PD-1 were observed in GL261 tumors where APM remained functional. This study demonstrates that one of the possible mechanisms of tumor resistance to immunotherapy is a dysfunctional APM and reveals a predictive potential of APM-related gene set expression for the personalization of dendritic cell vaccine and anti-PD-1 therapies in murine pre-treated tumors.

Nintedanib enhances the efficacy of PD-L1 blockade by upregulating MHC-I and PD-L1 expression in tumor cells.

Background: Immune checkpoint inhibitors (ICIs), such as programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1), have been widely applied in clinical and scientific research. Despite their effective antitumor effects in clinical tumor therapy, most tumors are still resistant to ICIs and long-term benefits are lacking. In addition, tumor patients complicated with interstitial lung disease limit the application of ICI therapy. Therefore, for these cases, there is an urgent need to develop new methods to relieve lung complications and enhance the efficacy of ICI therapy. Nintedanib, a potent triple angiokinase inhibitor approved for the treatment of progressive fibrotic interstitial lung disease. However, its immunotherapy synergy properties and mechanism are still pending further exploration.Methods: To explore the therapeutic potential of nintedanib and αPD-L1 combination therapy, MC38, LLC, and 4T1 tumor models were used to investigate antitumor and antimetastatic activities in vivo. An idiopathic pulmonary fibrosis-tumor bearing model was used to evaluate the effect of the synergy therapy on tumor model complicated with lung disease. Moreover, RNA-seq, immunohistochemistry, and flow cytometry were utilized to analyze the effect of combination treatment on the tumor microenvironment. The bioactivity following different treatments was determined by western blotting, CCK-8, and flow cytometry.Results: In this study, nintedanib and αPD-L1 synergy therapy exhibited significant antitumor, antimetastatic and anti-pulmonary fibrosis effects. Both in vitro and in vivo experiments revealed that these effects included promoting vessel normalization, increasing infiltration and activation of immune cells in tumors, enhancing the response of interferon-gamma, and activating the MHC class I-mediated antigen presentation process. Moreover, our results showed an increased expression of PD-L1 and promoted phosphorylation of STAT3 after nintedanib (1 µM) treatment.Conclusion: The combination of nintedanib and αPD-L1 increased ICI therapy responses, relieved lung complications and further activated the tumor immune microenvironment; thus, exhibiting a notable antitumor effect. Accordingly, the nintedanib synergy strategy is expected to be a promising candidate therapy for tumor patients complicated with interstitial lung disease in clinical practice.

EZH2 inhibition activates dsRNA-interferon axis stress and promotes response to PD-1 checkpoint blockade in NSCLC.

Lung cancer is the leading cause of cancer death and immunotherapy had been approved be a useful approach for NSCLC therapy. However, only part of the patients responds to checkpoint inhibitors. The EZH2, as a histone modification regulator, is overexpressed in NSCLC and negatively regulates the interferon-stimulated genes. Here, we demonstrate that EZH2 inhibition increases the double-strand RNA (dsRNA) level and then triggers the IFN pathway stress which is dependent on the pattern recognition receptors (TLR3, MDA5). The antigen presentation genes and PDL1 were also upregulated by inhibition of EZH2. Furthermore, in the immunocompetent LLC tumor model, the inhibition of EZH2 causes tumor regression and enhances the CD8+T cell infiltration. The EZH2 depletion triggers significant responses of the LLC mouse model to anti-PD1 therapy. This study identifies that inhibition of EZH2 promotes the dsRNA interferon driven antitumor immunity and enhances the anti-PD1 antitumor efficacy in NSCLC. These data suggest that EZH2 inhibition combined with anti-PD1/PDL1 is a promising lung cancer treatment strategy.

Anti-neoplastic pharmacophore benzophenone-1 coumarin (BP-1C) targets JAK2 to induce apoptosis in lung cancer.

Reigning of the abnormal gene activation associated with survival signalling in lung cancer leads to the anomalous growth and therapeutic failure. Targeting specific cell survival signalling like JAK2/STAT3 nexus has become a major focus of investigation to establish a target specific treatment. The 2-bromobenzoyl-4-methylphenoxy-acetyl hydra acetyl Coumarin (BP-1C), is new anti-neoplastic agent with apoptosis inducing capacity. The current study was aimed to develop antitumor phramacophore, BP-1C as JAK2 specific inhibitor against lung neoplastic progression. The study validates and identifies the molecular targets of BP-1C induced cell death. Cell based screening against multiple cancer cell lines identified, lung adenocarcinoma as its specific target through promotion of apoptosis. The BP-1C is able to induce, specific hall marks of apoptosis and there by conferring anti-neoplastic activity. Validation of its molecular mechanism, identified, BP-1C specifically targets JAK2Tyr1007/1008 phosphorylation, and inhibits its downstream STAT3Tyr705 signalling pathway to induce cell death. As a consequence, modulation in Akt/Src survival signal and altered expression of interwoven apoptotic genes were evident. The results were reproducible in an in-vivo LLC tumor model and in-ovo xenograft studies. The computational approaches viz, drug finger printing confers, BP-1C as novel class JAK2 inhibitor and molecular simulations studies assures its efficiency in binding with JAK2. Overall, BP-1C is a novel JAK2 inhibitor with experimental evidence and could be effectively developed into a promising drug for lung cancer treatment.

Neoantigen Dendritic Cell Vaccination Combined with Anti-CD38 and CpG Elicits Anti-Tumor Immunity against the Immune Checkpoint Therapy-Resistant Murine Lung Cancer Cell Line LLC1.

Simple SummaryDespite the substantial achievements to date, a significant proportion of patients still fail to benefit from immune-checkpoint therapies (ICT). The absence of T cell infiltration and insufficient immune recognition may account for the primary resistance to immune checkpoint therapy. The present study compared the ICT response of two murine lung cancer cell line models, ASB-XIV and LLC1. ASB-XIV tumors are inflamed and are sensitive to ICT, while non-inflamed LLC1 tumors are resistant. We employed in-depth tumor analysis, including whole-exome sequencing, RNA-sequencing, and flow cytometry, to reveal the molecular mechanisms of resistance to ICT, and sought strategies to promote inflammatory/immunogenic pathway activation and inhibit immunosuppressive factors present in LLC1 tumors. An appropriate vaccination strategy combining neoantigen peptide-pulsed DC with anti-CD38 antibody can render an ICT-resistant “cold” tumor susceptible to immune rejection via a mechanism involving neutralization of regulatory T cells. Thus, the future direction of ICT is combination immunotherapy.An important factor associated with primary resistance to immune-checkpoint therapies (ICT) is a “cold” tumor microenvironment (TME), characterized by the absence of T cell infiltration and a non-inflammatory milieu. Whole-exome and RNA sequencing to predict neoantigen expression was performed on the LLC1 cell line which forms “cold” tumors in mice. Dendritic cell (DC)-based vaccination strategies were developed using candidate neoantigen long peptides (LPs). A total of 2536 missense mutations were identified in LLC1 and of 132 candidate neoantigen short peptides, 25 were found to induce CD8+ T cell responses. However, they failed to inhibit LLC1 growth when incorporated into a cancer vaccine. In contrast, DCs pulsed with LPs induced CD4+ and CD8+ T cell responses and one of them, designated L82, delayed LLC1 growth in vivo. By RNA-Seq, CD38 was highly expressed by LLC1 tumor cells and, therefore, anti-CD38 antibody treatment was combined with L82-pulsed DC vaccination. This combination effectively suppressed tumor growth via a mechanism relying on decreased regulatory T cells in the tumor. This study demonstrated that an appropriate vaccination strategy combining neoantigen peptide-pulsed DC with anti-CD38 antibody can render an ICT-resistant “cold” tumor susceptible to immune rejection via a mechanism involving neutralization of regulatory T cells.