Colorectal cancer is a leading cause of cancer related deaths in the U.S., with African-Americans having higher incidence and mortality rates than Caucasian-Americans. Recent studies have demonstrated that anti-tumor cytotoxic T lymphocytes provide protection to patients with colon cancer while patients deficient in these responses have significantly worse prognosis. To determine if differences in cytotoxic immunity might play a role in racial disparities in colorectal cancer 258 microsatellite-stable colon tumors were examined for infiltrating immune biomarkers via immunohistochemistry. Descriptive summary statistics were calculated using two-sample Wilcoxon rank sum tests, while linear regression models with log-transformed data were used to assess differences in race and Pearson and Spearman correlations were used to correlate different biomarkers. The association between different biomarkers was also assessed using linear regression after adjusting for covariates. No significant differences were observed in CD8+ (p = 0.83), CD57+ (p = 0.55), and IL-17-expressing (p = 0.63) cell numbers within the tumor samples tested. When infiltration of granzyme B+ cells was analyzed, however, a significant difference was observed, with African Americans having lower infiltration of cells expressing this cytotoxic marker than Caucasians (p<0.01). Analysis of infiltrating granzyme B+ cells at the invasive borders of the tumor revealed an even greater difference by race (p<0.001). Taken together, the data presented suggest differences in anti-tumor immune cytotoxicity may be a contributing factor in the racial disparities observed in colorectal cancer.

Retrieval inhibition hypothesis of directed forgetting effects assumed TBF (to-be-forgotten) items were not retrieved intentionally, while selective rehearsal hypothesis assumed the memory representation of retrieved TBF (to-be-forgotten) items was weaker than TBR (to-be-remembered) items. Previous studies indicated that directed forgetting effects of item-cueing method resulted from selective rehearsal at encoding, but the mechanism of retrieval inhibition that affected directed forgetting of TBF (to-be-forgotten) items was not clear. Strategic retrieval is a control process allowing the selective retrieval of target information, which includes retrieval orientation and strategic recollection. Retrieval orientation via the comparison of tasks refers to the specific form of processing resulted by retrieval efforts. Strategic recollection is the type of strategies to recollect studied items for the retrieval success of targets. Using a “directed forgetting” paradigm combined with a memory exclusion task, our investigation of strategic retrieval in directed forgetting assisted to explore how retrieval inhibition played a role on directed forgetting effects. When TBF items were targeted, retrieval orientation showed more positive ERPs to new items, indicating that TBF items demanded more retrieval efforts. The results of strategic recollection indicated that: (a) when TBR items were retrieval targets, late parietal old/new effects were only evoked by TBR items but not TBF items, indicating the retrieval inhibition of TBF items; (b) when TBF items were retrieval targets, the late parietal old/new effect were evoked by both TBR items and TBF items, indicating that strategic retrieval could overcome retrieval inhibition of TBF items. These findings suggested the modulation of strategic retrieval on retrieval inhibition of directed forgetting, supporting that directed forgetting effects were not only caused by selective rehearsal, but also retrieval inhibition.

Advanced peritoneal carcinomatosis including high-grade ovarian cancer has poor prognoses and a poor response rate to current checkpoint inhibitor immunotherapies; thus, there is an unmet need for effective therapeutics that would provide benefit to these patients. Here we present the preclinical development of SENTI-101, a cell preparation of bone marrow-derived mesenchymal stromal (also known as stem) cells (MSC), which are engineered to express two potent immune-modulatory cytokines, IL12 and IL21. Intraperitoneal administration of SENTI-101 results in selective tumor-homing and localized and sustained cytokine production in murine models of peritoneal cancer. SENTI-101 has extended half-life, reduced systemic distribution, and improved antitumor activity when compared with recombinant cytokines, suggesting that it is more effective and has lower risk of systemic immunotoxicities. Treatment of tumor-bearing immune-competent mice with a murine surrogate of SENTI-101 (mSENTI-101) results in a potent and localized immune response consistent with increased number and activation of antigen presenting cells, T cells and B cells, which leads to antitumor response and memory-induced long-term immunity. Consistent with this mechanism of action, co-administration of mSENTI-101 with checkpoint inhibitors leads to synergistic improvement in antitumor response. Collectively, these data warrant potential clinical development of SENTI-101 for patients with peritoneal carcinomatosis and high-grade ovarian cancer. Graphical abstract: SENTI-101 schematic and mechanism of action SENTI-101 is a novel cell-based immunotherapeutic consisting of bone marrow–derived mesenchymal stromal cells (BM-MSC) engineered to express IL12 and IL21 intended for the treatment of peritoneal carcinomatosis including high-grade serous ovarian cancer. Upon intraperitoneal administration, SENTI-101 homes to peritoneal solid tumors and secretes IL12 and IL21 in a localized and sustained fashion. The expression of these two potent cytokines drives tumor infiltration and engagement of multiple components of the immune system: antigen-presenting cells, T cells, and B cells, resulting in durable antitumor immunity in preclinical models of cancer.

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Background: Colorectal cancer is the second leading cause of cancer deaths in the US. Genetic predisposition occurs in ∼30% of cases, suggesting that treatments with the ability to decrease lesion formation could significantly improve disease-free survival in potential patients with a strong family history. Genetically-engineered bacterial minicells (MC) are noninfectious, nano-sized bacterial particles derived from E. coli that can display immunomodulatory and antitumor activity in animal models of cancer. The expression of Invasin on the MC surface targets them to cancer-associated integrin heterodimers exposed on malignant cells, where they deliver Perfringolysin O, a unique tumorlytic bacterial toxin, that destroys these cells. We hypothesize that these MC can reduce colon lesions in genetically predisposed animals and that they have potential to be developed as a ‘probiotic’ therapy for individuals at high risk to develop colorectal cancer. Methods: The TS4CRE mouse line expresses the Cre recombinase specifically in the distal ileum and colon under the control of the fatty acid binding protein-4 promoter. The Apcfl-468 mouse line has lox-P recombination sites flanking exons 11-12 of the Apc gene. Cross-breeding results in a deletion in one allele of the Apc gene in a tissue-specific manner, resulting in lesion formation by 14-18 weeks of age. Cross-bred mice were treated intra-rectally with 1.5×109 MC or PBS, once a week for 6 doses, at either 8-13 or 14-19 weeks of age and harvested at various times after treatment is complete to evaluate tumor number and size. Tumor and tumor adjacent tissue is evaluated for immune biomarkers using qPCR and immunohistochemistry studies. Mann-Whitney (two-tailed) tests are used to determine statistical significance. Results: MC treatment during lesion development (14-19 weeks of age) significantly decreases tumor number (p = 0.007) and size (p = 0.03) in mice harvested at 6 months of age as compared to controls. Similarly, in animals treated from 8-13 weeks of age, when lesions are absent or only in the earliest stages, a significant decrease in tumor number (p = 0.04) was observed. MC can reduce tumor load through direct killing of developing lesions but they also have the potential to modulate immune responses. Since immune/inflammatory mediators are known to significantly contribute to tumor progression in colorectal cancer, decreased tumors in MC treated mice could be the result, at least in part, of a change in the immune/inflammatory environment of the colon. Chloracetate Esterase (CAE) staining for inflammatory granulocytes and mast cells showed that numbers of CAE+ cells in tumors and normal adjacent tissue from MC treated mice (at 14-19 weeks of age) were significantly lower than PBS-treated controls (p = 0.02). Moreover we saw that cells expressing CD11b, a biomarker for inflammatory, myeloid-lineage cells, were also significantly decreased in MC treated mice (p = 0.01). In addition, using qPCR we have determined that both the CD8 and IFNγ biomarkers were significantly increased in the tumor tissue of MC treated mice (p = 0.03 for each) while perforin and Tbet were increased in the tumor-adjacent tissue in these same mice (p = 0.04 for each), when compared to controls. Conclusions: Taken together, these data strongly suggest that MC reduce colon tumor burden in genetically predisposed mice, most likely both by directly eliminating tumor cells and changing the immune environment of the lesions. The immunomodulatory effects appear to consist of both increased anti-tumor immunity and decreased inflammatory cell infiltration. Future studies will continue to access the actions of these MC and the possibility that they can be developed as a ‘probiotic’ therapeutic approach against colorectal cancer in genetically predisposed individuals. Citation Format: Mengxi Tian, Mohammad W. Khan, Shea Grenier, Shingo Tsuji, Matthew A. Giacalone, Kathleen L. McGuire. Bacterial minicells decrease tumor development and modulate immunity in a mouse mouse model of colon cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B017.

<div>Abstract<p>Advanced peritoneal carcinomatosis including high-grade ovarian cancer has poor prognoses and a poor response rate to current checkpoint inhibitor immunotherapies; thus, there is an unmet need for effective therapeutics that would provide benefit to these patients. Here we present the preclinical development of SENTI-101, a cell preparation of bone marrow-derived mesenchymal stromal (also known as stem) cells (MSC), which are engineered to express two potent immune-modulatory cytokines, IL12 and IL21. Intraperitoneal administration of SENTI-101 results in selective tumor-homing and localized and sustained cytokine production in murine models of peritoneal cancer. SENTI-101 has extended half-life, reduced systemic distribution, and improved antitumor activity when compared with recombinant cytokines, suggesting that it is more effective and has lower risk of systemic immunotoxicities. Treatment of tumor-bearing immune-competent mice with a murine surrogate of SENTI-101 (mSENTI-101) results in a potent and localized immune response consistent with increased number and activation of antigen presenting cells, T cells and B cells, which leads to antitumor response and memory-induced long-term immunity. Consistent with this mechanism of action, co-administration of mSENTI-101 with checkpoint inhibitors leads to synergistic improvement in antitumor response. Collectively, these data warrant potential clinical development of SENTI-101 for patients with peritoneal carcinomatosis and high-grade ovarian cancer.</p><p>Graphical abstract: SENTI-101 schematic and mechanism of action</p><p>SENTI-101 is a novel cell-based immunotherapeutic consisting of bone marrow–derived mesenchymal stromal cells (BM-MSC) engineered to express IL12 and IL21 intended for the treatment of peritoneal carcinomatosis including high-grade serous ovarian cancer. Upon intraperitoneal administration, SENTI-101 homes to peritoneal solid tumors and secretes IL12 and IL21 in a localized and sustained fashion. The expression of these two potent cytokines drives tumor infiltration and engagement of multiple components of the immune system: antigen-presenting cells, T cells, and B cells, resulting in durable antitumor immunity in preclinical models of cancer.</p></div>

While immunotherapies based on single recombinant cytokines such as IL12 and IL21 have shown great promise in preclinical models of solid tumors, clinical translation has proven challenging due to limited mechanisms of action, narrow therapeutic windows upon systemic administration, and short half-lives resulting in poor pharmacokinetics and distribution. Thus, there is a need for tumor-localized cytokine therapies capable of driving sustained efficacy with a wide therapeutic window. SENTI-101 is a cell-based immunotherapy comprising allogeneic bone marrow-derived mesenchymal stromal cells (BM-MSCs) genetically modified to express IL12 and IL21. Consistent with prior studies, we demonstrated that SENTI-101 innately homes to disseminated tumors in the peritoneal cavity and induces durable anti-tumor responses and immune memory in various preclinical models of peritoneal tumors. In this study, we investigated the mechanisms of action of SENTI-101. Our results demonstrate that the IL12 and IL21 combination elicits pleiotropic and complementary effects that drive a multi-modal immune response across various steps of the cancer immunity cycle. Treatment of preclinical murine models of peritoneal tumors (e.g., CT26 and B16F10) with SENTI-101 significantly increased the local production of IFNg by more than 40-fold (p&lt;0.02). Concurrently, mice treated with SENTI-101 had significantly increased levels of cytokines and chemokines such as CXCL9 (p&lt;0.02), which have previously been associated with better prognosis and response to immunotherapy in multiple cancer types. We used flow cytometry and multiplexed IHC to characterize the immune landscape in response to SENTI-101. The number of antigen-presenting cells (F4/80negCD11c+MHC-II+CD103+) more than doubled in peritoneal lymph nodes 72 h after treatment with SENTI-101 (p=0.016). We also observed an increase in pSTAT1 positivity in the myeloid compartment, indicating a favorable immune phenotype. This change was accompanied by an increase in T-cell infiltrates into tumors (p=0.0003) that were in close proximity with B-cells and that were organized in tertiary lymphoid structures, which are known to correlate with improved prognosis in cancer patients. T-cell activation markers (CD38, CD25, IFNg, GranzymeB) also increased by more than 6 times (p=0.015) in the tumor microenvironment (TME) and peritoneal fluid after treatment with SENTI-101. In accordance with increased T-cell infiltration and activation in the TME, SENTI-101 showed a synergistic anti-tumor effect when combined with checkpoint inhibitor anti-PD1. Overall, our preclinical studies show that SENTI-101 modulates the tumor immune landscape via multiple complementary modes of action, resulting in long-term anti-tumor immunity. Furthermore, this work demonstrates the therapeutic potential of tumor-localized cell therapies armed with gene circuits expressing combinatorial immune effectors to trigger a multi-factorial anti-tumor response. Citation Format: Alba Gonzalez, Frances D. Liu, Archana Nagaraja, Alyssa Mullenix, Russell M. Gordley, Daniel O. Frimannsson, Anissa Benabbas, Chen-Ting Lee, Tiffany A. Truong, Allison Quach, Mengxi Tian, Rowena Martinez, Rishi Savur, Alyssa Perry-McNamara, Don-Hong Wang, Ori Maller, Dharini Iyer, Ashita Magal, Sravani Mangalampalli, Christina J. Huynh, Carmina C. Blanco, Jack T. Lin, Brian S. Garrison, Philip Lee, Timothy K. Lu, Gary Lee. SENTI-101, a novel genetically modified allogeneic cell product expressing IL12 and IL21, elicits a tumor-localized, robust, and multimodal immune response in preclinical models of solid tumors [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 4246.

<p>Supplementary Figure1 shows MSC homing to tumor and other organs in vivo using a bioluminescent reporter. Supplementary Figure 2. Characterization of tumor immune infiltrate of syngeneic preclinical intraperitoneal tumor models CT26-ip and B16F10-ip using IHC and flow cytometry. Supplementary Figure 3. Kaplan Meier Survival in B16F10-ip model comparing multiple combinations of effector cytokines expressed by MSCs. Supplementary Figure 4. Shows data supporting the construct optimization for SENTI-101 including promoter, orientation and signal sequence changes. Supplementary Figure 5. IL-21 functional validation using UO2S IL-21 receptor dimerization reporter assay. Supplementary Table 1. Shows the DNA sequence of SB00880, the lentiviral construct used to engineer SENTI-101. Supplementary Figure 6. Shows the SENTI-101 manufacturing process overview. Supplementary Figure 7. Shows supporting data for the study of pharmacokinetics and pharmacodynamics of SENTI-101. Supplementary Figure 8. Shows individual tumor burden for each mouse measured by bioluminescence imaging (BLI) after treatment with SENTI-101 or controls. Supplementary Figure 9. Percent change in mouse body weight after treatment. Supplementary Figure 10. Shows the controls and supporting data for the selective depletion of immune cell types. Supplementary Figure 11. Characterization of tumor infiltrating lymphocytes (TILs) in the B16F10-ip tumor model after treatment with mSENTI-101. Supplementary Figure 12. Anti-tumor immune response induced by SENTI-101 in CT-26ip model. Supplementary Figure 13. Shows individual tumor burden for each mouse measured by BLI after treatment with SENTI-101 and in combination with checkpoint inhibitor anti-PD-1.</p>