B16 Tumor-bearing Mice Research Articles

Combination therapy with oncolytic virus and T cells or mRNA vaccine amplifies antitumor effects

Antitumor therapies based on adoptively transferred T cells or oncolytic viruses have made significant progress in recent years, but the limited efficiency of their infiltration into solid tumors makes it difficult to achieve desired antitumor effects when used alone. In this study, an oncolytic virus (rVSV-LCMVG) that is not prone to induce virus-neutralizing antibodies was designed and combined with adoptively transferred T cells. By transforming the immunosuppressive tumor microenvironment into an immunosensitive one, in B16 tumor-bearing mice, combination therapy showed superior antitumor effects than monotherapy. This occurred whether the OV was administered intratumorally or intravenously. Combination therapy significantly increased cytokine and chemokine levels within tumors and recruited CD8+ T cells to the TME to trigger antitumor immune responses. Pretreatment with adoptively transferred T cells and subsequent oncolytic virotherapy sensitizes refractory tumors by boosting T-cell recruitment, down-regulating the expression of PD-1, and restoring effector T-cell function. To offer a combination therapy with greater translational value, mRNA vaccines were introduced to induce tumor-specific T cells instead of adoptively transferred T cells. The combination of OVs and mRNA vaccine also displays a significant reduction in tumor burden and prolonged survival. This study proposed a rational combination therapy of OVs with adoptive T-cell transfer or mRNA vaccines encoding tumor-associated antigens, in terms of synergistic efficacy and mechanism.

A novel CXCR4-targeted peptide for SPECT/CT imaging in tumor

As the most commonly expressed chemokine receptor in tumor cells, CXCR4 is associated with the development and metastasis of a variety of tumors, which has always been an important target for tumor diagnosis and treatment. We present a series of peptides designed to target CXCR4 and determine their affinity values (Kd) for human CXCR4 proteins using surface ion resonance technology. Despite the significant variations in their affinities, our results demonstrate the feasibility of our peptide engineering strategy. Among them, YQ-X-9 (Gly-Gly-Pro-D-Tyr-Arg-D-Val-Gly-Arg-D-2Nal-Pro-NH2) had the highest affinity (Kd=530 nM) compared to the positive control AMD3100 (Kd=240 nM). Furthermore, YQ-X-9 was selected as the target and 99 mTc was functionalized by HYNIC to form a specific targeting probe (99 mTc-HYNIC-YQ-X-9). Additionally, we established a xenograft mouse model of B16 melanoma cells for in vivo SPECT/CT imaging, resulting in evident tumor uptake, excellent imaging efficacy, and high contrast. And in vivo distribution experiments confirmed that in B16 tumor-bearing mice, tumors had significant uptake, reaching 2.4% ID/g. Therefore, 99 mTc-HYNIC-YQ-X-9 may serve as a promising peptide-based probe for clinical imaging of CXCR4 overexpressed tumors.’

The use of xenogenic testicular antigens for induction of antitumor reactions

Testicular antigens (TAGs) are normally expressed only by cells of testicular and placental tissues. Human immune system is tolerant to TAG, but if the integrity of the testicular membranes is disrupted, these antigens, entering the bloodstream, induce autoimmune reactions for eliminating them from the body. In malignancy, TAGs begin to be expressed by tumor cells of the liver, breast, pancreas, intestine, and lung. Immunological recognition of these AGs leads to autoimmune reactions against these AGs, i.e. antitumor reactions in the body. We used xenogenic TAGs derived from ram testis to increase TAG immunogenicity. The use of ram TAGs is justified by the fact that TAGs are evolutionarily conserved molecules and there is a high degree of homology between human and animal TAGs.The purpose of the study was to evaluate the lifespan of tumor-bearing mice and parameters of cellular immunity in various options for immunizing mice with ram TAGs.Material and Methods. C57BL/6 mice were used. The efficacy of therapeutic or prophylactic vaccination with xenogenic TAGs was studied by changing lifespan of B16 and LLC tumor-bearing mice. Formation of immune responses was evaluated by proliferative ability of splenocytes to respond to vaccination and control AGs and by their production of IFN-gamma and IL-10.Results. In the LLC carcinoma model with a preventive vaccination option, the lifespan of mice with syngeneic vaccination did not differ from the tumor control; the lifespan of mice with xenogeneic vaccination increased by 60%. In therapeutic vaccination option, no significant differences in lifespan of vaccinated mice were found. A significant increase in the proliferative activity of splenocytes in response to tumor AGs was found in both LLC- and B16 tumor-bearing mice previously vaccinated with xenogenic TAGs. The increased IFN-gamma production by splenocytes was observed in B16 and LLC tumorbearing mice with xenogeneic vaccination. The IFN-gamma production by splenocytes in tumor-bearing mice with syngeneic vaccination was not increased. A significant decrease in IL-10 production was noted in mice with xenogeneic vaccination.

CHEK2 deficiency increase the response to PD-1 inhibitors by affecting the tumor immune microenvironment

Immune checkpoint blockade (ICB) therapy has improved treatment effects in multiple cancers. Gene mutations in the DNA damage repair pathway (DDR) may cause genomic instability and may relate to the efficacy of ICB. Checkpoint kinase 2 (CHEK2) and polymerase epsilon (POLE) are important genes in the DDR. In this study, we aimed to study the impact of CHEK2 deficiency mutations on the response to ICB. We found that tumors with CHEK2 mutations had a significantly higher tumor mutational burden (TMB) compared to those with CHEK2-WT in a pancancer database. We noted that CHEK2 deficiency mutations potentiated the anti-tumor effect of anti-PD-1 therapy in MC38 and B16 tumor-bearing mice with the decrease of tumor volume and tumor weight after anti-PD-1 treatment. Mechanistically, CHEK2 deficiency tumors were with the increased cytotoxic CD8+ T-cell infiltration, especially cytotoxic CD8+ T cells, and modulated the tumor-immune microenvironment with an upregulated immune inflammatory pathway and antigen presentation pathway after anti-PD-1 treatment. Furthermore, murine models with POLE mutations confirmed that CHEK2 deficiency shaped similar mutational and immune landscapes as POLE mutations after anti-PD-1 treatment. Taken together, our results demonstrated that CHEK2 deficiency mutations may increase the response to ICB (eg. anti-PD-1) by influencing the tumor immune microenvironment. This indicated that CHEK2 deficiency mutations were a potentially predictive biomarker and CHEK2 deficiency may potentiate response to immunotherapy.

Abstract LB566: Class 1 HDAC inhibition induces antitumor immunity by NF-kB-mediated enhanced metabolic fitness and generation of unique effector function enriched memory CD8 T cell subtype

Abstract Histone/protein deacetylases (HDACs) are a class of enzymes that regulate gene expression and are involved in regulating the transcription process as well as regulating fundamental cellular processes such as proliferation, differentiation and development. Class I HDACs (which includes HDAC1, 2, 3, and 8) contribute in epigenetic regulations of T cell tolerance. Under anergic T cell condition, HDAC1 and HDAC2 are involved in the histone deacetylation. However, the role of HDACs in CD8 T cell function and differentiation is not well understood. Therefore, we aimed to understand the effect of HDAC inhibition (HDACi) on CD8 T cells phenotype and functionality by using Entinostat (ENT), a class I HDAC (HDAC1 and 3) inhibitor. We found that ENT induces strong immune-mediated anti-tumor effects in TC-1 and B16 tumor-bearing mice. Mechanistically, ENT modulated the tumor microenvironment through an increased infiltration of CD8 T cells, enhanced CD8 T cell antigen-specificity and CD8 T cell functionality represented by interferon gamma (IFN-γ) and granzyme B (GrB) secretion. Our in vitro and in vivo data also indicate that ENT enhances central memory phenotype (CD62L+, CD44+) of CD8 T cells. We also found that ENT-treated CD8 T cells have distinct metabolic phenotype compared to activated CD8 T cells. Specifically, these cells relied more on amino acid metabolism while, unexpectedly, they had lower fatty acid uptake with enhanced mitochondrial potential (TMRM). Interestingly, ENT-induced central memory CD8 T cells also showed increased mitochondrial potential as well as reliance on amino acid metabolism. Therefore, ENT treatment induced memory cells with a unique phenotype different from the classical memory CD8 T cells with enhanced effector functions and reliance on distinct metabolic pathways. Furthermore, ENT-treated CD8 T cells have higher peaks at H3K27Ac site compared to the control. Since these peaks are mostly associated with genes such as IFN-γ, GrB, Pim1, EOMES, and IL-2, the known canonical target genes of NF-κB in CD8 T cells, we further dissected the role of NF-κB in these ENT-mediated effects. First, motif analysis showed that NF-κB binding motif is enriched in differential H3K27Ac sites associated with HDACi. Next, flow cytometry and immunofluorescence analysis showed that although ENT does not increase total NF-κB levels in CD8 T cells, it enhances its binding to the chromatin due to enhanced gene accessibility. This research highlights the significance of epigenetic modulation in enhancing the immune-mediated anti-tumor effects and provide fundamental understanding of the mechanism of action of HDAC inhibition in CD8 T cells. Citation Format: Nour Shobaki, Pankaj Gaur, Rahul Nadre, Vivek Verma, Peter Ordentlich, Lei Wang, Nazli Jafarzadeh, Wael Traboulsi, Mikayel Mkrtichyan, Seema Gupta, Samir N. Khleif. Class 1 HDAC inhibition induces antitumor immunity by NF-kB-mediated enhanced metabolic fitness and generation of unique effector function enriched memory CD8 T cell subtype [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 LB566.

Abstract 6150: Potential role of CD47 in T cell exhaustion program

Abstract Multiple suppressive mechanisms within the tumor microenvironment (TME) are capable of blunting anti-tumor T cell responses. These include engagement of inhibitory receptors expressed in tumor-associated, exhausted CD8 T cells, such as programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), 2B4 (also known as CD244), and T cell immunoreceptor with Ig and ITIM domains (TIGIT). While immune checkpoint blockade therapies aimed at reversing the dysfunctional state of tumor-associated T cells have demonstrated clinical effectiveness, not all cancer patients achieve long-term disease control. This is due, at least in part, to the refractory nature of what are categorized as terminally exhausted CD8 T cells to be reinvigorated by, for example, PD-1/PD-L1 blockade. As CD8 T cell exhaustion (or dysfunction) is a major therapeutic challenge, gaps in our understanding of cellular and molecular mechanisms underlying the T cell exhaustion (or dysfunction) program in cancer warrant further study of pathways that program T cells toward exhaustion (or dysfunction). Through comprehensive immune profiling of tumor-infiltrating T lymphocytes (TILs), we found that CD47 expression in CD8 TILs isolated from melanoma patients significantly correlates with expression of several checkpoint inhibitory molecules (e.g., TIM-3, PD-1 and LAG-3). Additionally, our re-analysis of single cell data from melanoma patients revealed that terminally exhausted T cells (Tex) and TCF7hi Tex precursor cells exhibit high levels of CD47 transcripts, suggesting phenotypic association of CD47 with T cell exhaustion. We confirmed our observations in murine B16-F10 melanoma where CD47 expression is significantly upregulated in exhausted CD8 TILs. We also show that CD47 functions as a negative regulator for T cell proliferation and function during T cell priming. To address the role of CD47 during the development of CD8 T cell exhaustion/dysfunction in cancer, we performed adoptive T cell transfer of the naïve-sorted Cd47+/+ (WT) and Cd47+/- (Het) antigen specific Pmel-1 CD8 T cells (but not Cd47-deficient Pmel-1 CD8 T cells as they would be subject to innate immune clearance) into B16 tumor-bearing mice and found that Cd47-Het Pmel-1 CD8 TILs, as compared to the Cd47-WT Pmel-1 CD8 TILs, exhibit less expression of exhaustion-related genes (e.g. Pdcd1, Lag3 and Tox), and increased expression of genes associated with T cell activation and proliferation (e.g. Mki67, Lck, Cd69, Gzma, Gzmk). We further confirmed that thrombospondin-1 (TSP-1), as an extracellular matrix protein and a ligand of CD47, contributes to driving the differentiation of CD8 T cells toward exhaustion. Our data highlight for the first time the potential of extracellular matrix protein TSP-1 in programming CD8 T cell exhaustion in cancer through its interaction with CD47 expressed on CD8 T cells. Citation Format: Chien-Huan Weng, Fadi Samaan, Sadna Budhu, Levi Mangarin, Sébastien Monette, Cailian Liu, Stephane Pourpe, Linda Hamadene, Hong Zhong, Xia Yang, David Schroder, Roberta Zappasodi, Pamela Holland, Jedd D. Wolchok, Taha Merghoub. Potential role of CD47 in T cell exhaustion program [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 6150.

Abstract 4217: MEKi in combination with anti-OX40 generates metabolically fit effector CD8+ T cells, enhances stem cell-like memory CD8+ T cells, and leads to strong anti-tumor activity

Abstract In the tumor microenvironment (TME), T cell exhaustion and short persistence of effector cells are crucial factors that limit the efficacy of endogenously generated or adoptively transferred effector cells. Regenerative stem-cell memory CD62LhiCD44lowSca1+ CD8+ CD8+ T cells (TSCM) persist longer and produce cells with more vital effector functions. OX40, a member of TNFRSF4, has been shown to promote the expansion and proliferation of activated effector T cells, leading to the generation of robust anti-tumor responses and immune memory. However, treatment with anti-OX40 as a single therapeutic modality has not generated desirable clinical outcomes. A combination of immune modulators has been found to enhance the therapeutic efficacy synergistically. Recently, we reported that inhibition of MEK1/2 using selumetinib (AZD6244) leads to a significant increase in the numbers of CD8+ TSCM cells with self-renewability, enhanced multipotency and proliferative capacity, leading to potent anti-tumor effects. Therefore, here we combined selumetinib with anti-OX40 to investigate if enhanced immune-mediated anti-tumor responses could be obtained. Using three mouse tumor models (TC1, B16, and CT26), we found that combination of MEKi with anti-OX40 enhances the anti-tumor activity by increasing the expansion of total and antigen-specific CD8+ T cells in the TME. These CD8+ T cells have higher functional activity as seen by increased Granzyme B and INF-γ production. We also found similar results after adoptive cell therapy (ACT) where infusion of MEKi-treated cells in B16 tumor-bearing mice synergized with anti-OX40 agonist treatment. Interestingly, anti-OX40 treatment of MEKi-treated CD8+ T cells enhanced the metabolic fitness as demonstrated by low mitochondrial potential and enhanced Oxygen Consumption Rates (OCR), Spare Respiratory Capacity (SRC), and Extra-cellular Acidification Rates (ECAR) compared to anti-OX40 treatment alone. Furthermore, OX40 agonist significantly enhanced the longevity of MEKi-induced CD8+ TSCM cells. In summary, our data provide a novel strategy to utilize MEKi to enhance the efficacy of ACT and combination immunotherapy using anti-OX40 agonist. Citation Format: Pankaj Gaur, Vivek Verma, Mays Alreem Elayyan, Zainab Ramlaoui, Simon T. Barry, Viia E. Valge-Archer, Paul D. Smith, Seema Gupta, Samir N. Khleif. MEKi in combination with anti-OX40 generates metabolically fit effector CD8+ T cells, enhances stem cell-like memory CD8+ T cells, and leads to strong anti-tumor activity [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 4217.

Immunotherapy combining tumor and endothelium cell lysis with immune enforcement by recombinant MIP-3α Newcastle disease virus in a vessel-targeting liposome enhances antitumor immunity

BackgroundSeveral agents for oncolytic immunotherapy have been approved for clinical use, but monotherapy is modest for most oncolytic agents. The combination of several therapeutic strategies through recombinant and nanotechnology to...

A pH-sensitive supramolecular nanosystem with chlorin e6 and triptolide co-delivery for chemo-photodynamic combination therapy

The combination of Ce6, an acknowledged photosensitizer, and TPL, a natural anticancer agent, has been demonstrated as a useful strategy to reinforce the tumor growth suppression, as well as decrease the systemic side effects compared with their monotherapy. However, in view of the optimal chemo-photodynamic combination efficiency, there is still short of the feasible nanovehicle to steadily co-deliver Ce6 and TPL, and stimuli-responsively burst release drugs in tumor site. Herein, we described the synergistic antitumor performance of a pH-sensitive supramolecular nanosystem, mediated by the host–guest complexing between β-CD and acid pH-responsive amphiphilic co-polymer mPEG-PBAE-mPEG, showing the shell–core structural micelles with the tight β-CD layer coating. Both Ce6 and TPL were facilely co-loaded into the spherical supramolecular NPs (TPL+Ce6/NPs) by one-step nanoprecipitation method, with an ideal particle size (156.0 nm), acid pH-responsive drug release profile, and enhanced cellular internalization capacity. In view of the combination benefit of photodynamic therapy and chemotherapy, as well as co-encapsulation in the fabricated pH-sensitive supramolecular NPs, TPL+Ce6/NPs exhibited significant efficacy to suppress cellular proliferation, boost ROS level, lower MMP, and promote cellular apoptosis in vitro. Particularly, fluorescence imaging revealed that TPL+Ce6/NPs preferentially accumulated in the tumor tissue area, with higher intensity than that of free Ce6. As expected, upon 650-nm laser irradiation, TPL+Ce6/NPs exhibited a cascade of amplified synergistic chemo-photodynamic therapeutic benefits to suppress tumor progression in both hepatoma H22 tumor-bearing mice and B16 tumor-bearing mice. More importantly, lower systemic toxicity was found in the tumor-bearing mice treated with TPL+Ce6/NPs. Overall, the designed supramolecular TPL+Ce6/NPs provided a promising alternative approach for chemo-photodynamic therapy in tumor treatment.

Safety levels of systemic IL-12 induced by cDNA expression as a cancer therapeutic.

Aim: The aim of this work is to utilize a gene expression procedure to safely express systemic IL-12 and evaluate its effects in mouse tumor models. Materials & methods: Secondary lymphoid organs and tumors from EL4 and B16 tumor-bearing mice were analyzed by supervised and unsupervised methods. Results: IL-12 cDNA induced systemic IL-12 protein levels lower than the tolerated dose in patients. Control of tumor growth was observed in subcutaneous B16 and EL4 tumors. Systemic IL-12 expression induced a higher frequency of both total tumor-infiltrated CD45+ cells and proliferative IFN-γ+CD8+T cells along with a lower frequency of CD4+FOXP3+ and CD11b+Gr-1+ cells. Conclusion: This approach characterizes the systemic effects of IL-12, helping to improvetreatment of metastases or solid tumors.

Abstract 1845: Enhancing the efficacy of antitumor antibodies with immune checkpoint inhibitors and targeted therapy in melanoma

Abstract The therapeutic outcomes of current strategies in melanoma treatment have greatly improved within the last decade. However, the persistent relatively low success rate of current standard-of-care strategies underscores the urgent need for improvement of these therapeutic approaches. The use of specific antitumor antibodies recently transformed the cancer therapeutics landscape with successes such as anti-HER2 in breast cancer, and anti-EGFR in HNSCC and colorectal cancer. However, antitumor antibodies have not been developed for the treatment of melanoma. Here, using mouse preclinical models of melanoma (both solid tumor and lung metastasis), we found that the therapeutic efficacy of the specific antitumor antibody TA99 (anti-TYRP1) is significantly enhanced by immune checkpoint blockade. This was associated with a greater CD8+, CD8+/Foxp3+, NK1.1+ and dendritic cell infiltrate, suggestive of an increased anti-tumor immune response. Further, MEK inhibition (MEKi) increased the expression of melanosomal antigens in B16 and YUMM mouse melanoma cell lines in vitro. We next combined TA99 and MEKi to treat B16 melanoma in vivo and observed a synergistic reduction in tumor growth. Treatment of mice bearing YUMM melanoma (BRAF mutant) with TA99/MEKi resulted in the complete elimination of tumors in the majority of animals, and produced durable responses. Moreover, we found a synergistic effect when B16 tumor-bearing mice were treated with TA99 combined with MEKi and immune checkpoint blockade (anti-PD1 and anti-CTLA4). Our findings suggest that MEKi induced an increased expression of tumor-associated antigens, which, in combination with antitumor antibodies, generated a robust adaptive antitumor response that was sustained by immune checkpoint inhibition therapy. These improved therapeutic effects of specific antitumor antibodies by current standard-of-care therapeutics have potential near-term translation to the clinic, in light of fully humanized anti-TYRP1 antibodies currently in clinical development. Citation Format: Rolando Pérez-Lorenzo, Stephanie O. Erjavec, Raphael Clynes, Angela M. Christiano. Enhancing the efficacy of antitumor antibodies with immune checkpoint inhibitors and targeted therapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1845.

B16 melanoma control by anti-PD-L1 requires CD8+ T cells and NK cells: application of anti-PD-L1 Abs and Trp2 peptide vaccines

ABSTRACT Anti-programmed death ligand 1 (PD-L1) therapy has been beneficial in treating patients with certain cancers. Here, we tested whether anti-PD-L1 therapy is effective for controlling different types of tumors using animal models of TC-1, MC38 and B16. We found that, despite PD-L1 expression, anti-PD-L1 therapy showed little and some antitumor activity in the TC-1 and MC38 models. However, anti-PD-L1 therapy exhibited a more dramatic antitumor effect in the B16 model. This difference in antitumor responses was likely associated with the CD8 + T cell infiltration status of tumor tissues. In the B16 model, CD8 + T cells and to a lesser degree NK cells were found to be responsible for the antitumor response of anti-PD-L1 therapy, as determined by immune cell subset depletion. In particular, CD8 + T cells from B16-bearing mice produced an IFN-γ in response to B16 cells and citrate phosphate buffer-treated B16 cell peptide elutes but not to an immunodominant class I epitope, Trp2180-188, suggesting that CD8 + T cells that recognize neoantigens were induced in B16 tumor-bearing mice and then reactivated by anti-PD-L1 for tumor control. When B16 tumor-bearing mice were treated with anti-PD-L1 in combination with Trp2180-188 peptide vaccines, they displayed significantly more tumor control than either single therapy. Taken together, these studies show that B16 melanomas are more effectively controlled through reactivation of tumor-infiltrating lymphocytes by anti-PD-L1 therapy. Moreover, combined therapy using anti-PD-L1 and Trp2 peptide vaccines is more beneficial for controlling B16 melanomas through reactivation of neoantigen-specific CD8 + T cells and induction of Trp2-specific CD8 + T cells.

Voluntary wheel running can lead to modulation of immune checkpoint molecule expression

Background Exercise and physical activity (PA) are associated with reduced tumor growth and enhanced intra-tumoral immune cell infiltration in mice. We aimed to investigate the role of PA achieved by voluntary wheel running in promoting the immunogenic profile across several murine tumor models, and to explore the potential of checkpoint blockade and PA in the form of voluntary wheel running as combination therapy. Material and methods The experiments were performed with C57BL/6 mice bearing subcutaneous tumors while having access to running wheels in their cages, where key immunoregulatory molecules expressed in the tumor tissue were measured by qPCR. Furthermore, we tested the hypothesis that wheel running combined with PD-L1 -or PD-1 inhibitor treatment could lead to an additive effect on tumor growth in mice bearing B16 melanoma tumors. Results Wheel running increased immune checkpoint expression (PD-1, PD-L1, PD-L2, CD28, B7.1 and B7.2) in B16 tumor-bearing mice, while induction of only PD-L2 was found in E0771 breast cancer and Lewis Lung Cancer. In studies combining voluntary wheel running with PD-1 -and PD-L1 inhibitors we found significant effects of wheel running on attenuating B16 melanoma tumor growth, in line with previous studies. We did, however, not find an additive effect of combining either of the two immunotherapeutic treatments with access to running wheels. Conclusion B16 tumors displayed upregulated expression of immune regulatory molecules and decreased tumor growth in response to PA. However, combining PA with PD-1 or PD-L1 blockade did not lead to a further augmented inhibition of tumor growth.

Nonpeptidic quinazolinone derivatives as dual nucleotide-binding oligomerization domain-like receptor 1/2 antagonists for adjuvant cancer chemotherapy.

Nucleotide-binding oligomerization domain-containing protein 1 and 2 (NOD1/2) receptors are potential immune checkpoints. In this article, a quinazolinone derivative (36b) as a NOD1/2 dual antagonist was identified that significantly sensitizes B16 tumor-bearing mice to paclitaxel treatment by inhibiting both nuclear factor κB (NF-κB) and mitogen-activated protein kinase inflammatory signaling that mediated by NOD1/2.

Abstract 2665: Immune cell modulation of pulsed focused ultrasound in murine melanoma and breast cancer models

Abstract Major therapeutic advances in the field of tumor immunotherapy are hindered by the ability to overcome the immunosuppressive tumor microenvironment (TME), which prevents the immune system from mounting an efficient anti-tumor response. Pulsed focused ultrasound (pFUS) studies has shown some promise as an immunomodulatory strategy for altering TME. This study evaluated the temporal changes following non-ablative pFUS on TME immune cell profiles in spleen (Sp), lymph nodes (LN), and tumors (B16 melanoma and 4T1 mammary carcinoma). Murine B16 and 4T1 cells were subcutaneously, bilaterally implanted into C57BL/6J and BALB/c mice (n=10 tumors/time-point), respectively. Ultrasound guided pFUS (VIFU 2000, Alpinion) was administered at 1 MHz, at 6 MPa PNP to 5 mm tumors. Entire tumor volume was sonicated with a 2 mm spacing between points using a 10 ms pulse length, 10% duty cycle and a pulse repetition frequency of 10 Hz. At days 1, 3, and 5 post-sonication, Sp, LN and tumors were harvested, fixed and processed for flow cytometry (FACS) analysis of immune cell populations [cytotoxic (Tcyt), helper (Th), and regulatory (Treg) T cells, natural killer (NK), dendritic cells (DC), F4/80+ macrophages (M1 and M2), myeloid-derived suppressor cells (MDSC)] and immune checkpoint markers (CTLA-4, PD-1 and PD-L1). The immune response to pFUS differed between the B16 and 4T1 tumor bearing mice. B16 mice at days 1, 3 and 5 post-sonication revealed a 3-4 fold increase in Tcyt, Th, NK and M1 macrophage activity in tumor, which peaked at day 3, and decreased by day 5, compared to control tumors. At day 5, a shift of cell populations toward LN, with increased Treg, Tcyt, NK, F4/80, M1 and M2 activity was observed. 4T1 mice showed high levels of Tcyt in Sp on day 1, and NK cells on day 3 in LN. Interestingly, these cell populations decreased in Sp and LN by day 5, with 3-4 fold increase in 4T1 tumors, along with increasing macrophage and DC activity. Despite the differences in both tumor types, results suggest that pFUS altered the TME by inducing anti-tumor responses by activating both innate (macrophages, NK cells) and adaptive (Tcyt, Th cells) arms of immunity. This study reveals the differences in immune cell profiles following sonication between the two tumor types and underscores the importance of not generalizing the effect of pFUS on the TME. This analysis also suggests that the global changes to the immune cell profile represent a shift from anti-inflammatory, immunosuppressive TME towards an anti-tumor TME. Optimizing pFUS parameters to enhance immune cell infiltration, would presumably lead to slowing tumor growth rates and when coupled with immune checkpoint inhibits could serve as neoadjuvant approach in treating malignancy. Citation Format: Parwathy Chandran, Gadi Cohen, Scott R. Burks, Joseph A. Frank. Immune cell modulation of pulsed focused ultrasound in murine melanoma and breast cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2665.

A recombinant oncolytic Newcastle virus expressing MIP-3α promotes systemic antitumor immunity

BackgroundThe oncolytic Newcastle disease virus (NDV) is inherently able to trigger the lysis of tumor cells and induce the immunogenic cell death (ICD) of tumor cells and is also an...

Abstract A10: Constitutive HIF activity enhances T-cell antitumor immunity and memory T-cell formation in the murine adoptive cell therapy model

Abstract Purpose: To determine if unrestrained hypoxia inducible factor (HIF) activity in CD8 T cells improves antitumor response. Methods: CD8 T cells were isolated from von Hippel Lindau (VHL)-deficient (VHL KO) mice, which have unrepressed HIF1 and HIF2 activity, as well as from VHL/HIF1/HIF2 triple-deficient (TKO) and WT mice. Following in vitro activation, T cells were transferred to melanoma (B16) tumor-bearing mice and monitored for tumor progression and survival. In other studies, activated VHL KO and TKO CD8 cells were mixed with WT CD8 cells and transferred to B16 or adenocarcinoma (MC38) tumor-bearing mice; after 7-10 days, donor T cells were analyzed from tumors, spleens, and draining lymph nodes (LN). Results: Single transfer of VHL KO CD8 T cells to B16 tumor-bearing mice resulted in delayed tumor growth and prolonged survival compared to transfer of CD8 T cells from TKO or WT donors. Co-transfer of VHL KO cells with WT cells into B16 tumor-bearing mice resulted in superior accumulation of VHL KO cells in all tissues including tumors. In contrast, mixed transfer of TKO cells with WT cells to B16 tumor-bearing mice resulted in poorer accumulation of TKO cells in all tissues including tumors. VHL KO tumor-infiltrating lymphocytes (TILs) had superior expression of tissue resident memory (TRM) phenotype markers CD103 and CD69 compared to WT and TKO TILs. Similarly in the MC38 tumor model, VHL KO cells showed improved accumulation in all tissues compared to WT cells. Conclusions: Here we demonstrate that VHL deletion in CD8 T cells results in superior efficacy in delaying tumor growth and improving recipient survival, greater accumulation in multiple solid tumor models, and increased development of the TRM phenotype. Further, TKO recipients demonstrated increased tumor growth, reduced survival, reduced TIL numbers, and absence of TRM phenotype following adoptive transfer, confirming the HIF dependency of the VHL-deletion phenotype. Taken together, these data demonstrate that T cells with constitutive HIF activity provide a more robust antitumor response. Therefore, manipulation of HIF regulation could offer new tools for enhanced T-cell efficacy in adoptive cellular therapy for solid tumors. Citation Format: Colette R. Lauhan, Deborah Witherden, Ilkka Liikanan, Ananda Goldrath. Constitutive HIF activity enhances T-cell antitumor immunity and memory T-cell formation in the murine adoptive cell therapy model [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A10.

Phosphatidylserine targeting antibody enhances anti-tumor activity of adoptive cell therapies in a mouse melanoma model

Abstract Adoptive cell therapy has emerged as a viable strategy to treat cancer. T cells that recognize tumor antigens can be reinvigorated ex-vivo or autologous T cells can be genetically modified to express anti-tumor T cell receptors (TCRs) or chimeric antigen receptors (CARs). However, once re-infused into patients, these tumor specific T cells are subjected to immunosuppressive signals within the tumor. A critical immune checkpoint within tumors is phosphatidylserine (PS), a phospholipid that is exposed on apoptotic cells and tumor cells. Innate cells exposed to PS secrete suppressive cytokines that can significantly impair the function of tumor specific T cells. Antibodies that target PS can reactivate anti-tumor immunity by reducing the number of MDSCs in tumors and promoting the maturation of functional APCs. Our lab has shown that the mouse chimeric version of PS Targeting monoclonal antibody Bavituximab (1N11), in combination with transgenic CD4+ T cells that recognize melanoma antigen Trp1, can regress advanced melanoma tumors in mice. Here, we demonstrate a 2nd generation CAR T cell, that binds Trp1 on the surface of B16 melanoma, in combination with 1N11 can improve anti-tumor activity and survival in B16 tumor bearing mice. Flow cytometry analysis of immune responses in the tumor of mice treated with tumor specific T cells and 1N11 shows a decrease in M2 macrophages and FoxP3+ regulatory T cells. These findings highlight that diminishing suppressive mechanisms locally with PS targeting can enhance the efficacy of transgenic TCR and CAR T cells to improve the outcome in patients with advanced-stage melanoma. Our studies may inform the design of clinical trials combining PS Targeting antibodies with CAR T cell therapy in solid tumors.

Abstract B83: RNA-nanoparticles for immunotherapy-resistant head and neck squamous cell carcinoma

Abstract Introduction: Immunotherapy is promising for patients with treatment-refractory head and neck squamous cell carcinoma (HNSCC). However, >80% of patients do not respond to checkpoint inhibitors, suggesting that the highly immunosuppressive tumor immune microenvironment of HNSCC may play a role in treatment resistance. Our group has developed a novel treatment platform utilizing clinically translatable RNA-nanoparticles (NPs) to remodel the systemic and intratumoral HNSCC microenvironment in favor of an activated immunologic milieu. Objective: The purpose of this study is to examine the feasibility, safety and immunologic activity of systemic/intratumoral administration of RNA NPs for HNSCC. Materials and Methods: RNA-NPs were prepared by complexing negatively charged mRNA with nanoliposomes. These complexes were injected into naive and B16 or MOC1/MOC2 (murine oral cavity squamous cell carcinoma cell lines) tumor bearing C57Bl/6 mice. In vitro transfection experiments were performed in human HNSCC cell lines. Results: Human HNSCC cell lines were efficiently transfected with RNA NPs in vitro. MOC1/MOC2 tumors were efficiently transfected with RNA NPs in vivo. RNA-NPs activate dendritic cells (DCs) in the peripheral and intratumoral microenviroment of naive and B16 tumor bearing mice, and mediate antitumor efficacy in a preclinical in vivo murine model for HNSCC. RNA-NPs mediate their effects through interferon-alpha released from plasmacytoid DCs. We have successfully scaled up production of RNA-NPs for translation into human clinical trials and demonstrated safety of RNA-NPs in acute/chronic murine toxicity studies based on CBCs, electrolytes, organ function tests, and end-organ H&E histology. Conclusions: RNA-NPs safely reprogram the peripheral and intratumoral immune microenvironments, unlocking antitumor activity. We have successfully generated RNA-NPs, providing a renewable resource that can be made readily available for all HNSCC patients. FDA-IND enabling studies are currently under way in preparation for first-in-human trials for checkpoint refractory HNSCC. Citation Format: Natalie L. Silver, Rekha R. Garg, Kristianna M. Fredenberg, Hector R. Mendez, Duane A. Mitchell, Elias J. Sayour. RNA-nanoparticles for immunotherapy-resistant head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B83.

Abstract B2: Coexpression of effector T cell-associated chemokine receptors defines T-cell subsets with different roles in antitumor response and toxicity of dual checkpoint blockade

Abstract Dual checkpoint blockade has yielded impressive clinical responses in cancer treatment, but only a subset of patients responds and immune-related adverse events (irAE) are common, frequently leading to treatment discontinuation. Both CCR5 and CXCR3 expression are associated with effector T-cell trafficking and favorable outcomes in various tumors, but only CXCR3 was shown to exhibit a nonredundant role for cytotoxic T-cell trafficking across tumor vessels, while CCR5 has been implicated in recruitment of regulatory T cells to the tumor microenvironment. Here, we explored the coexpression of T cell-associated chemokine receptors in human melanoma and studied the effect of genetic and pharmacologic manipulation of CCR5 on PD-1/CTLA-4 (P1C4) blockade efficacy and toxicity observed in B16 tumor-bearing mice. We found that in human melanoma CCR5 and CXCR3 expression characterizes different T-cell subsets based on their coexpression with other chemokine receptors at the single-cell level. These subsets exhibit distinct phenotypic and functional characteristics in blood and tumor; CXCR3 indicated less differentiated T cells with greater proliferative potential while CCR5 expression was more abundant on terminal effector cells. In addition, subsets expressing only CXCR3 were increased in patients who received checkpoint blockade while subsets that express CCR5 were unaffected by the treatment. In murine melanoma, dual checkpoint inhibition using PD-1/CTLA-4 blockade significantly increased CXCR3, but not CCR5 expression on T cells. Moreover, a murine model of PD-1/CTLA-4 blockade-induced irAE showed high levels of CCR5+ T cells in toxicity target organs. When PD-1/CTLA-4 inhibitors were administered to either CCR5-KO mice or WT mice treated with the CCR5 antagonist maraviroc, there was a significant decrease in tumor growth and improved survival compared to WT mice receiving P1C4 alone. Together these results reveal distinct, potentially opposite roles for CCR5 and CXCR3 subsets specifically in the setting of dual checkpoint blockade, that can be redirected to improve efficacy and reduce toxicity observed in melanoma. Citation Format: Rodney J.M. Macedo Gonzales, David Harle, Ximi K. Wang, Alexandra Matschiner, Ran Reshef. Coexpression of effector T cell-associated chemokine receptors defines T-cell subsets with different roles in antitumor response and toxicity of dual checkpoint blockade [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B2.