Mouse syngeneic tumor models are widely used tools to demonstrate activity of novel anti-cancer immunotherapies. Despite their widespread use, a comprehensive view of their tumor-immune compositions ...and their relevance to human tumors has only begun to emerge. We propose each model possesses a unique tumor-immune infiltrate profile that can be probed with immunotherapies to inform on anti-tumor mechanisms and treatment strategies in human tumors with similar profiles. In support of this endeavor, we characterized the tumor microenvironment of four commonly used models and demonstrate they encompass a range of immunogenicities, from highly immune infiltrated RENCA tumors to poorly infiltrated B16F10 tumors. Tumor cell lines for each model exhibit different intrinsic factors in vitro that likely influence immune infiltration upon subcutaneous implantation. Similarly, solid tumors in vivo for each model are unique, each enriched in distinct features ranging from pathogen response elements to antigen presentation machinery. As RENCA tumors progress in size, all major T cell populations diminish while myeloid-derived suppressor cells become more enriched, possibly driving immune suppression and tumor progression. In CT26 tumors, CD8 T cells paradoxically increase in density yet are restrained as tumor volume increases. Finally, immunotherapy treatment across these different tumor-immune landscapes segregate into responders and non-responders based on features partially dependent on pre-existing immune infiltrates. Overall, these studies provide an important resource to enhance our translation of syngeneic models to human tumors. Future mechanistic studies paired with this resource will help identify responsive patient populations and improve strategies where immunotherapies are predicted to be ineffective.
Tumor cell adaptation to hypoxic stress is an important determinant of malignant progression. While much emphasis has been placed on the role of HIF‐1 in this context, the role of additional ...mechanisms has not been adequately explored. Here we demonstrate that cells cultured under hypoxic/anoxic conditions and transformed cells in hypoxic areas of tumors activate a translational control program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum (ER) stress. Inactivation of ISR signaling by mutations in the ER kinase PERK and the translation initiation factor eIF2α or by a dominant‐negative PERK impairs cell survival under extreme hypoxia. Tumors derived from these mutant cell lines are smaller and exhibit higher levels of apoptosis in hypoxic areas compared to tumors with an intact ISR. Moreover, expression of the ISR targets ATF4 and CHOP was noted in hypoxic areas of human tumor biopsy samples. Collectively, these findings demonstrate that activation of the ISR is required for tumor cell adaptation to hypoxia, and suggest that this pathway is an attractive target for antitumor modalities.
Purpose
The presence and functional competence of intratumoral CD8
+
T cells is often a barometer for successful immunotherapeutic responses in cancer. Despite this understanding and the extensive ...number of clinical-stage immunotherapies focused on potentiation (co-stimulation) or rescue (checkpoint blockade) of CD8
+
T cell antitumor activity, dynamic biomarker strategies are often lacking. To help fill this gap, immuno-PET nuclear imaging has emerged as a powerful tool for
in vivo
molecular imaging of antibody targeting. Here, we took advantage of immuno-PET imaging using
89
Zr-IAB42M1-14, anti-mouse CD8 minibody, to characterize CD8
+
T-cell tumor infiltration dynamics following ICOS (inducible T-cell co-stimulator) agonist antibody treatment alone and in combination with PD-1 blocking antibody in a model of mammary carcinoma.
Procedures.
Female BALB/c mice with established EMT6 tumors received 10 µg, IP of either IgG control antibodies, ICOS agonist monotherapy, or ICOS/PD-1 combination therapy on days 0, 3, 5, 7, 9, 10, or 14. Imaging was performed at 24 and 48 h post IV dose of
89
Zr IAB42M1-14. In addition to
89
Zr-IAB42M1-14 uptake in tumor and tumor-draining lymph node (TDLN), 3D radiomic features were extracted from PET/CT images to identify treatment effects. Imaging mass cytometry (IMC) and immunohistochemistry (IHC) was performed at end of study.
Results
89
Zr-IAB42M1-14 uptake in the tumor was observed by day 11 and was preceded by an increase in the TDLN as early as day 4. The spatial distribution of
89
Zr-IAB42M1-14 was more uniform in the drug treated
vs.
control tumors, which had spatially distinct tracer uptake in the periphery relative to the core of the tumor. IMC analysis showed an increased percentage of cytotoxic T cells in the ICOS monotherapy and ICOS/PD-1 combination group compared to IgG controls. Additionally, temporal radiomics analysis demonstrated early predictiveness of imaging features.
Conclusion
To our knowledge, this is the first detailed description of the use of a novel immune-PET imaging technique to assess the kinetics of CD8
+
T-cell infiltration into tumor and lymphoid tissues following ICOS agonist and PD-1 blocking antibody therapy. By demonstrating the capacity for increased spatial and temporal resolution of CD8
+
T-cell infiltration across tumors and lymphoid tissues, these observations underscore the widespread potential clinical utility of non-invasive PET imaging for T-cell-based immunotherapy in cancer.
We previously identified four novel cDNA fragments related to human esophageal cancer. One of the fragments was named esophageal cancer related gene 2 (ECRG2). We report here the molecular cloning, ...sequencing, and expression of the ECRG2 gene. The ECRG2 cDNA comprises a 258 bp nucleotide sequence which encodes for 85 amino acids with a predicted molecular weight of 9.2 kDa. Analysis of the protein sequence reveals the presence at the N terminus of a signal peptide followed by 56 amino acids with a significant degree of sequence similarity with the conserved Kazal domain which characterizes the serine protease inhibitor family. Pulse-chase experiments showed that ECRG2 protein was detected in both cell lysates and culture medium, indicating that the ECRG2 protein was extracellularly secreted after the post-translational cleavage. In vitro uPA/plasmin activity analysis showed the secreted ECRG2 protein inhibited the uPA/plasmin activity, indicating that ECRG2 may be a novel serine protease inhibitor. Northern blot analysis revealed the presence of the major band corresponding to a size of 569 kb throughout the fetal skin, thymus, esophagus, brain, lung, heart, stomach, liver, spleen, colon, kidney, testis, muscle, cholecyst tissues and adult esophageal mucosa, brain, thyroid tissue and mouth epithelia. However, ECRG2 gene was significantly down-regulated in primary esophageal cancer tissues. Taken together, these results indicate that ECRG2 is a novel member of the Kazal-type serine protease inhibitor family and may function as a tumor suppressor gene regulating the protease cascades during carcinogenesis and migration/invasion of esophageal cancer.
Abstract
In recent years, antibody-based immunotherapies targeting immune checkpoint proteins cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death receptor-1 (PD-1), have revolutionized the ...treatment of cancer. Despite durable clinical responses in many indications, a significant number of patients fail to yield meaningful benefit, emphasizing the need to identify novel and/or complementary therapeutic interventions (e.g., alternative immune checkpoints, cytotoxic agents, etc.). Inducible T cell Co-Stimulator (ICOS/CD278) is a receptor belonging to the CD28/CTLA-4 superfamily that is predominantly expressed on T cells shortly after T cell receptor (TCR) activation. A growing body of non-clinical and clinical evidence supports the concept of antibody-mediated induction of ICOS signaling as a means to promote durable antitumor responses, particularly in collaboration with other checkpoint immunotherapies. To support ongoing and future clinical studies for feladilimab (GSK3359609), a non-depleting IgG4 ICOS agonist antibody currently being evaluated in pivotal clinical trials, we conducted a series of in vivo studies using a rodent surrogate (7E.17G9 mouse mIgG1; analogous non-depleting Fc) in ICOS-sensitive (EMT6 breast carcinoma) and ICOS-insensitive (CT26 colon carcinoma) tumor models. Female BALB/c mice with pre-established EMT6 tumors, exhibited partial responses to GSK'609 surrogate/tool antibody (7E.17G9 mIgG1), with ~20% (range 0-40%) of mice demonstrating tumor clearance. Concurrent administration of anti-PD-1 (RMP1-14 rat rIgG2a) and CTLA-4 (9D9 mIgG2b) dramatically improved tumor growth inhibition and survival. Notably, a triplet of TIM-3 blockade (RMT3-23 rIgG2a) in concert with ICOS and PD-1 mAbs further enhanced the survival of EMT6 tumor-bearing mice, suggesting complementary therapeutic mechanisms. In addition to combination with immune checkpoint blockade, we also explored the potential of ICOS co-stimulation with different focal irradiation (FRT) doses and frequencies in both EMT6 and CT26 tumor models. Intriguingly, we observed dramatic differences in response to concurrent FRT and ICOS mAb depending on the tumor model and associated FRT dose and frequency. Further, we identified a FRT dose threshold required for ICOS agonist appreciable combination responses, enabling exploration of additional immunotherapeutic combinations on top of ICOS/FRT. Collectively, these data support the combination of ICOS co-stimulation with different checkpoint immunotherapies (doublets and triplets), several of which are currently being evaluated in clinical studies. Moreover, these data demonstrate potential of ICOS co-stimulation with cytotoxic strategies, such as FRT - providing various dosing considerations and a framework future therapeutic intervention strategies.
Citation Format: Jeremy D. Waight, Meixia Bi, Chris Hopson, Sapna Yadavilli, Kenneth W. Hance, Marc Ballas, Axel Hoos. ICOS co-stimulation in combination immune checkpoint blockade and/or dose-optimized focal irradiation results in enhanced tumor control 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 1849.
The proto-oncogene c-Myc paradoxically activates both proliferation and apoptosis. In the pathogenic state, c-Myc-induced apoptosis is bypassed via a critical, yet poorly understood escape mechanism ...that promotes cellular transformation and tumorigenesis. The accumulation of unfolded proteins in the ER initiates a cellular stress program termed the unfolded protein response (UPR) to support cell survival. Analysis of spontaneous mouse and human lymphomas demonstrated significantly higher levels of UPR activation compared with normal tissues. Using multiple genetic models, we demonstrated that c-Myc and N-Myc activated the PERK/eIF2α/ATF4 arm of the UPR, leading to increased cell survival via the induction of cytoprotective autophagy. Inhibition of PERK significantly reduced Myc-induced autophagy, colony formation, and tumor formation. Moreover, pharmacologic or genetic inhibition of autophagy resulted in increased Myc-dependent apoptosis. Mechanistically, we demonstrated an important link between Myc-dependent increases in protein synthesis and UPR activation. Specifically, by employing a mouse minute (L24+/-) mutant, which resulted in wild-type levels of protein synthesis and attenuation of Myc-induced lymphomagenesis, we showed that Myc-induced UPR activation was reversed. Our findings establish a role for UPR as an enhancer of c-Myc-induced transformation and suggest that UPR inhibition may be particularly effective against malignancies characterized by c-Myc overexpression.
Gastrin is a known growth/differentiation factor for the gastric mucosa. Its effects are likely mediated by the induction of heparin-binding epidermal-like growth factor (HB-EGF), a member of the EGF ...family of growth factors that is expressed by gastric parietal cells. In this study, we investigated the regulation of the HB-EGF promoter by gastrin in a human gastric cancer cell line. Serial human HB-EGF promoter-luciferase reporter deletion constructs and heterologous promoter constructs were transfected into AGS-E cells and stimulated with gastrin (10(-7) M) with or without various signal transduction inhibitors. EMSA were also performed. Gastrin stimulation resulted in a fivefold increase in HB-EGF-luciferase activity. The cis-acting element mediating gastrin responsiveness was mapped to the -69 to -58 region of the HB-EGF promoter. Gastrin stimulation was PKC dependent and at least partially mediated by activation of the EGF receptor.
Abstract
Unprecedented rates of durable clinical responses have been observed for antibody-based therapeutics targeting immune checkpoint proteins such as cytotoxic T lymphocyte antigen-4 (CTLA-4) or ...programmed death receptor-1 (PD-1). Nonetheless, a significant number of patients fail to respond, highlighting the need for alternative and complementary immunotherapeutic interventions for cancer. Inducible T-cell costimulator (ICOS/CD278) is a CD28 superfamily receptor that is predominantly expressed on effector and cytotoxic T cells shortly after T cell receptor (TCR) activation. In addition to promotion of T cell and B cell collaboration, ICOS signaling (via ICOS ligand ICOSL or antibody-based crosslinking) has been shown to improve T cell antitumor activity, survival, durable tumor rejection. To support the clinical development of GSK3359609 (a hIgG4 ICOS agonist antibody) several in vivo combination studies were conducted using a tool anti-mouse ICOS agonist antibody (clone 7E.17G9). Analogous with the Fc-based characteristics of a human IgG4 antibody, the anti-mouse ICOS tool antibody exhibited significant antitumor activity when grafted on a murine IgG1 (or Fc-reduced) backbone. Using an in vivo models of breast cancer (EMT6, BALB/c), the murinized ICOS tool antibody demonstrated potent antitumor activity alone (tumor growth inhibition TGI of 40-60%) in combination with immune checkpoint inhibitors (ICI) targeting PD-1 and CTLA-4 (TGI of 100% and 90%, respectively). Antitumor activity was associated with increased total T cell infiltration into the tumor, a higher tumor CD8/Treg cell ratio, and broad increases in T cell activation markers (e.g. CD25, CD69, Granzyme B, perforin, and PD-1). The cancer immunity cycle posits that multiple points of intervention exist for cancer immunotherapy, many of which should be considered in order to identify complementary therapeutic strategies. With this in mind, and consistent with ICI, antitumor activity was observed in combination with innate immune modulators (e.g. TLR4 agonist) and chemo/cytotoxic (e.g. carboplatin and paclitaxel) therapies in different syngeneic tumor models. Altogether these data underscore the broad utility of ICOS agonist antibodies as a reliable combination partner for cancer immunotherapy.
Citation Format: Jeremy D. Waight, Meixia Bi, David Kilian, Christopher Hopson, Shu-Yun Zhang, Sara Brett, Sapna Yadavilli, Tianqian Zhang, Hong Shi, Kenneth W. Hance, Marc Ballas, Axel Hoos. Non-clinical tumor models reveal broad combination potential of ICOS agonist antibodies 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 2220.
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