Cell Cytolytic Activity Research Articles

Abstract PS11-27: A phase I/II clinical trial of EDP1503 with pembrolizumab for triple-negative breast cancer

Abstract Background: Systemic immunity can be regulated via transient interactions of non-colonizing bacteria and immune cells in the small intestinal mucosa. The potential to harness these interactions for IO treatment was first described by Sivan in 2015. EDP1503 is a non-colonizing preparation of a single strain of Bifidobacterium animalis lactis that, when administered orally, in preclinical models, invokes an anti-tumor immune response by activation of innate and adaptive immune mechanisms. The pleiotropic effects of EDP1503 are initiated in the small intestine and mediated by interactions with multiple pattern-recognition receptors inducing a proinflammatory signature in human PBMCs, activating CD8 and NK cell IFNγ production and cytolytic activity. Preclinically, EDP1503 inhibits tumor growth as a monotherapy and in combination with anti-PD-1/L1. Single agent PD-1/L1 therapies have showed limited clinical benefit in previously treated triple-negative breast cancer (TNBC) patients. Here, we report the safety, tolerability, and efficacy of EDP1503 in combination with pembrolizumab in patients with TNBC (NCT03775850). Methods: EDP1503-101 is an open label Phase 1/2 study of EDP1503 in combination with pembrolizumab. TNBC subjects who had received ≥ 1 treatment regimen for metastatic disease were enrolled. Prior anti-PD-1/L1 treatment was permitted. Subjects initially receive 14 days of EDP1503 monotherapy orally twice a day (b.i.d) and then EDP1503 b.i.d. in combination with pembrolizumab 200 mg iv every 3 weeks. The initial 3 subjects received 2 capsules of EDP1503 b.i.d. All other subjects received 4 capsules b.i.d. Safety and tolerability were assessed using CTCAE v5.0. Evidence of anti-tumor activity was based on investigator-assessed objective response (OR), by RECIST v1.1 and iRECIST, and disease control rate defined as OR and/or stable disease (SD) after 4 cycles of therapy. Paired biopsies at baseline and day 14 were used to establish PD-L1 status and investigate pharmacodynamic biomarker changes. Results: As of 1 October 2020, 15 TNBC subjects had been treated (median age of 52, median of 2 prior lines of therapy and ECOG of 0-1). Additional subjects are being recruited, up to a maximum of 30. EDP1503 was well-tolerated as monotherapy and in combination with pembrolizumab. 53% of subjects experienced treatment-related adverse events (TRAEs). Most common TRAEs were GI-related: abdominal distension (20%), decreased appetite (20%), diarrhea (13%), flatulence (13%). No Grade 4-5 TRAEs were observed. 1 Grade 3 TRAE (diarrhea) leading to treatment discontinuation was reported. In 12 subjects receiving the 4 capsules b.i.d dose, 2 partial responses (PR) and 1 SD were observed, giving an objective response rate (ORR) of 18% and a disease control rate (DCR) of 27% in evaluable patients (n=11). Both responders had tumor burden reductions of >65% by RECIST. One responder had 4 prior lines of therapy for metastatic disease, including a PD-L1 inhibitor combination, has a PD-L1 combined positive score (CPS) of 30 and has been on study treatment for 192 days with a 66% reduction in target lesions. The only residual lesion is 6 mm which is PET-negative. The other responder had 2 prior lines of therapy for metastatic disease, was checkpoint inhibitor naive, has a PD-L1 CPS of 2 and remained on study treatment for 275 days with a 73% reduction in target lesions before discontinuing due to treatment-related AEs. The patient with SD had also previously relapsed on a PD-L1 inhibitor and was on study treatment for 226 days before progressing. Conclusions: EDP1503 administered with pembrolizumab is safe and well-tolerated with no Grade 4-5 TRAEs or SAEs. Clinical benefit was observed in a subset of TNBC patients treated with the combination of EDP1503 and pembrolizumab. This study is continuing to recruit TNBC patients at the high dose of EDP1503. Citation Format: Loise Francisco-Anderson, Shamira Shariffudin, Humphrey Gardner, Peter Sandy, Michael Goldberg, Shubhra Kashyap, Mary Abdou, Maria Sizova, Valeria Kravitz, Holly Ponichtera, Mark Carlson, Shannon Argueta, Chris Davitt, Pooja Parameswaran, Michael Chisamore, Mark Bodmer, Duncan McHale. A phase I/II clinical trial of EDP1503 with pembrolizumab for triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-27.

Using Elevated Cholesterol Synthesis as a Prognostic Marker in Wilms’ Tumor: A Bioinformatic Analysis

Background Wilms tumor is the most common renal malignancy of children. Identifying factors that could predict the prognosis of patients with Wilms tumor is clinically meaningful. Many studies found tumors with elevated cholesterol synthesis that are featured with dismal prognosis. Even in some clinical trials, people with excessive dietary cholesterol intake and high plasma low-density lipoprotein levels are observed to have increased risk for cancer. However, the role of cholesterol biosynthesis in Wilms tumor has not yet been well clarified. Methods RNA sequencing transcriptome data and all corresponding clinicopathological information used in our study were downloaded from the TARGET database. High-throughput sequencing (Fragments Per Kilobase of transcript per Million fragments mapped) data sets of 130 tumor samples and 6 normal samples were obtained for further analysis. Results Wilms tumor samples with higher activity of cholesterol synthesis are characterized with worse overall survival (P < 0.05). In addition, Wilms tumor samples with mitigated activity of cholesterol synthesis are featured with better dendritic cell (DC) function and cytolytic activity (P < 0.05). Furthermore, we constructed a prognosis model based on differential expressed cholesterol synthesis-related genes (DECSG), which could predict the OS of patients with Wilms tumor accurately. KEGG and GO analysis of differential expressed genes between tumor samples with high and low cholesterol synthesis indicated that DECSGs are highly enriched in “mitosis nuclear division,” “nuclear division,” “chromosome segregation,” “cell cycle,” “Spliceosome,” and “RNA transport.” Conclusions In conclusion, our study reported increased cholesterol synthesis in Wilms tumor predicts a worse prognosis and mitigated cytolytic activity, DC function, and MHC I signature in the tumor microenvironment. We also constructed a prognosis model for predicting the OS of patients with good accuracy, which is promising in clinical translation. Future studies should focus on the detailed mechanism that caused increasing cholesterol which promotes tumor progression and undermines patients' survival.

Dynamics of the NK cell cytolytic activity in acute myocardial infarction: the role of circulating immune complexes

NK cells play an important role in the pathogenesis of atherosclerosis, using their cytotoxic mechanisms that promote the initiation, progression, and rupture of atherosclerotic plaques that leads to acute myocardial infarction. However the data about their cytolytic activity in post-infarction period remain controversial. The aim of this work was to study the dynamics of changes in the NK cell cytolytic activity within three weeks after acute myocardial infarction in connection with the level of circulating immune complexes in the peripheral blood. Material and methods. 16 healthy donors and 35 patients after the development of acute myocardial infarction (AMI) on the 1st, 7th and 21st days was studied for the NK cell cytolytic activity of the mononuclear fraction of peripheral blood, evaluated by the cytolysis of erythroleukemia K-562 cells in vitro and levels of serum circulating immune complexes (CIC) using the method of precipitation with polyethylene glycol-6000. Results and discussion. In the most patients, AMI led to functional anergy of NK cells, but in the smaller part of patients, the increase in their cytotoxicity was observed. The post-infarction period was characterized by an increased content of CIC, the level of which positively correlated with the development of functional anergy of NK cells. Possible mechanisms for the development of both functional anergy and an increase in NK cell activity in the post-infarction period are discussed. Keywords: аcute myocardial infarction, NK cells, circulating immune complexes.

A bispecific T cell engager targeting Glypican-1 redirects T cell cytolytic activity to kill prostate cancer cells

BackgroundGlypican-1 is a heparan sulfate proteoglycan that is overexpressed in prostate cancer (PCa), and a variety of solid tumors. Importantly, expression is restricted in normal tissue, making it an ideal tumor targeting antigen. Since there is clinical and preclinical evidence of the efficacy of Bispecific T cell Engager (BiTE) therapy in PCa, we sought to produce and test the efficacy of a GPC-1 targeted BiTE construct based on the Miltuximab® sequence. Miltuximab® is a clinical stage anti-GPC-1 antibody that has proven safe in first in human trials.MethodsThe single chain variable fragment (scFv) of Miltuximab® and the CD3 binding sequence of Blinatumomab were combined in a standard BiTE format. Binding of the construct to immobilised recombinant CD3 and GPC-1 antigens was assessed by ELISA and BiaCore, and binding to cell surface-expressed antigens was measured by flow cytometry. The ability of MIL-38-CD3 to activate T cells was assessed using in vitro co-culture assays with tumour cell lines of varying GPC-1 expression by measurement of CD69 and CD25 expression, before cytolytic activity was assessed in a similar co-culture. The release of inflammatory cytokines from T cells was measured by ELISA and expression of PD-1 on the T cell surface was measured by flow cytometry.ResultsBinding activity of MIL-38-CD3 to both cell surface-expressed and immobilised recombinant GPC-1 and CD3 was retained. MIL-38-CD3 was able to mediate the activation of peripheral blood T cells from healthy individuals, resulting in the release of inflammatory cytokines TNF and IFN-g. Activation was reliant on GPC-1 expression as MIL-38-CD3 mediated only low level T cell activation in the presence of C3 cells (constitutively low GPC-1 expression). Activated T cells were redirected to lyse PCa cell lines PC3 and DU-145 (GPC-1 moderate or high expression, respectively) but could not kill GPC-1 negative Raji cells. The expression of PD-1 was up-regulated on the surface of MIL-38-CD3 activated T cells, suggesting potential for synergy with checkpoint inhibition.ConclusionsThis study reports preclinical findings into the efficacy of targeting GPC-1 in PCa with BiTE construct MIL-38-CD3. We show the specificity and efficacy of the construct, supporting its further preclinical development.

Chronic shift-lag promotes NK cell ageing and impairs immunosurveillance in mice by decreasing the expression of CD122.

Long‐term subjection to shift work increases the risk of cancer. The purpose of the present study was to explore the mechanism by which chronic circadian disruption impairs natural killer (NK) cell immunosurveillance. Mice were subjected to light‐dark reverse every 4 days for 12 weeks to disrupt normal circadian rhythm. NK cell development and function were evaluated by flow cytometry. The mRNA and protein levels of period 1 (per1) and per2 were suppressed, while circadian locomotor output cycle kaput (CLOCK) was increased in the shifted mice, indicating successful generation of the circadian rhythm disruption mouse model. Chronic shift‐lag promoted NK cell ageing, which is likely due to the reduction in Ly49 family receptor expression in shifted NK. We further studied the effects of circadian rhythm disruption on NK cell function. Chronic shift‐lag inhibited NK cell secretion of granular CD107a and interferon gamma. Moreover, chronic shift‐lag attenuated the clearance of MHC‐I–deficient tumour cells by NK cells in vivo and promoted lung metastasis of B16F10 melanomas. Furthermore, chronic shift‐lag reduced NK cell killing function, which may be due to the suppression of Eomes transcription factor expression, which inhibiting the transcription of CD122. In conclusion, our findings suggest that chronic circadian disruption attenuates NK cell cytolytic activity by decreasing the expression of CD122.

RAB11FIP5-Deficient Mice Exhibit Cytokine-Related Transcriptomic Signatures.

Rab11 recycling endosomes are involved in immunological synaptic functions, but the roles of Rab11 family–interacting protein 5 (Rab11Fip5), one of the Rab11 effectors, in the immune system remain obscure. Our previous study demonstrated that RAB11FIP5 transcripts are significantly elevated in PBMCs from HIV-1–infected individuals, making broadly HIV-1–neutralizing Abs compared with those without broadly neutralizing Abs; however, the role of Rab11FiP5 in immune functions remains unclear. In this study, a RAB11FIP5 gene knockout (RAB11FIP5−/−) mouse model was employed to study the role of Rab11Fip5 in immune responses. RAB11FIP5−/− mice exhibited no perturbation in lymphoid tissue cell subsets, and Rab11Fip5 was not required for serum Ab induction following HIV-1 envelope immunization, Ab transcytosis to mucosal sites, or survival after influenza challenge. However, differences were observed in multiple transcripts, including cytokine genes, in lymphocyte subsets from envelope-immunized RAB11FIP5−/− versus control mice. These included alterations in several genes in NK cells that mirrored observations in NKs from HIV-infected humans expressing less RAB11FIP5, although Rab11Fip5 was dispensable for NK cell cytolytic activity. Notably, immunized RAB11FIP5−/− mice had lower IL4 expression in CD4+ T follicular helper cells and showed lower TNF expression in CD8+ T cells. Likewise, TNF-α production by human CD8+ T cells correlated with PBMC RAB11FIP5 expression. These observations in RAB11FIP5−/− mice suggest a role for Rab11Fip5 in regulating cytokine responses.

Impaired natural killer cell counts and cytolytic activity in patients with severe COVID-19

AbstractThe global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–driven coronavirus disease 2019 (COVID-19) has caused unprecedented human death and has seriously threatened the global economy. Early data suggest a surge in proinflammatory cytokines in patients with severe COVID-19, which has been associated with poor outcomes. We recently postulated that the inflammatory response in patients with severe COVID-19 disease is not inhibited by natural killer (NK) cells, resulting in a “cytokine storm.” Here, we assessed the NK-cell functional activity and the associated cytokines and soluble mediators in hospitalized COVID-19 patients. Significantly impaired NK-cell counts and cytolytic activity were observed in COVID-19 patients when compared with healthy controls. Also, cytokines like interleukin 12 (IL12), IL15, and IL21 that are important for NK-cell activity were not detected systematically. Serum concentrations of soluble CD25 (sCD25)/soluble IL2 receptor α (sIL2-Rα) were significantly elevated and were inversely correlated with the percentage of NK cells. Impaired NK-cell cytolytic activity together with other laboratory trends including elevated sCD25 were consistent with a hyperinflammatory state in keeping with macrophage-activation syndrome. Our findings suggest that impaired counts and cytolytic activity of NK cells are important characteristics of severe COVID-19 and can potentially facilitate strategies for immunomodulatory therapies.

Abstract 6604: Drug responsive domain regulation of IL15-engineered T cells provides pharmacological control over antigen-independent cell expansion

Abstract Engineered T cell therapies have been remarkably successful in the treatment of B cell malignancies, yet lack of control over these “living drugs” can lead to significant toxicities or limited efficacy. One particular challenge is the achievement of durable anti-tumor responses because the reduction of tumor burden results in reduced antigen stimulation and therefore reduced antigen-dependent T cell expansion. Interleukin-15 (IL15) drives T and NK cell expansion and persistence in an antigen-independent manner, however unregulated expression of this cytokine may compromise the safety and efficacy of cellular immunotherapy. To address these issues, we engineered T cells with a pharmacologically controllable, membrane-bound IL15 that supports antigen-independent T cell expansion and has the potential to reduce the safety risks associated with continuous exposure to soluble IL15. Our approach utilizes Drug Responsive Domains (DRDs) which are fully human protein domains that are inherently unstable in the cell but are reversibly stabilized when bound to specific FDA-approved drugs. Fusion of a DRD to a protein of interest confers drug-dependent, reversible regulation of protein expression and function. We developed a DRD based on the carbonic anhydrase 2 (CA2) protein, which is stabilized in the presence of the FDA-approved drug acetazolamide (ACZ). Upon gene transfer of membrane-bound IL15 fused to a CA2 DRD, regulated IL15 expression on T cells was detected only in the presence of ACZ. In the absence of ACZ, the level of IL15 detected on the surface of gene-modified T cells is not substantially different from the level detected on untransduced T cells. ACZ treatment of gene-modified T cells increases surface IL15 expression in a dose-dependent manner. We observed prolonged survival and up to 15-fold expansion of IL15-CA2 DRD-transduced T cells in the absence of supplemental cytokines or antigen stimulation. In contrast, vehicle-treated IL15-CA2 DRD modified T cells and untransduced T cells did not survive or expand in vitro. Importantly, both IL15-CA2-transduced T cells and unengineered, co-infused NK cells survived and persisted significantly more in vivo in ACZ-treated but not vehicle-treated non-tumor-bearing NSG mice. Our results demonstrate that a novel membrane-bound IL15-CA2 fusion protein coupled with ACZ treatment induces antigen-independent T cell expansion and augments bystander NK cell persistence. Regulatable IL15 expression has significant implications for both T and NK cell therapies by providing more durable cell expansion and cytolytic activity in vivo, and thus the potential to significantly reduce cell dosing while maintaining clinical efficacy in patients. Citation Format: Steven Shamah, Kutlu Elpek, Tucker Ezell, Michelle Fleury, Michael Gallo, Jennifer Gori, Scott Heller, Mara Inniss, Meghan Langley, Grace Olinger, Celeste Richardson, Karen Tran, Dhruv Sethi, Dexue Sun, Vipin Suri. Drug responsive domain regulation of IL15-engineered T cells provides pharmacological control over antigen-independent cell expansion [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 6604.

Abstract 1607: Pro-cancerous immune profile in primary breast tumors affects the timing and type of breast cancer recurrence

Abstract Background: Tumor infiltrating lymphocytes (TILs) are immune cells that have migrated to the tumor tissue and the local microenvironment. Recently, evidence has emerged the presence of TILs affects the clinical outcomes of breast cancer patients. We hypothesize that immune cell infiltration profile of the primary tumors affects the timing and type of breast cancer recurrence. We evaluated the relationship between the timing and type of cancer recurrence and clinical factors, gene expression profiles, and immune cell infiltration profile utilizing two published large cohorts, TCGA and METABRIC. Methods: 308 primary BCs in TCGA were analyzed and categorized as: recurrence ≤2 years (Early), between 2-5 years (Mid), recurrence &amp;gt;5 years (Late), and no recurrence &amp;gt;5 years (Survivors). 1,727 primary BCs in METABRIC were analyzed and categorized similarly: Early, Mid, Late, and Survivors, and further subdivided according to the type of recurrence (distant or local recurrence) Results: We found that Early tumors demonstrated more aggressive clinical characteristics such as larger tumor, more lymph node metastases, higher pathological grades, higher Stages, and negative estrogen and progesterone receptors, compared with Survivors. On the other hand, no clinical characteristics of Late tumors were significantly different from Survivors, which implicate that clinical characteristics cannot distinguish late recurrence from Survivors. Utilizing pre-ranked gene set enrichment analyses, we showed that primary tumors of Survivors were associated with anti-cancer signaling such as interferon-α/-γ response and TNF-α signaling, compared with all recurrence groups. Furthermore, we found that host defense immunity (leukocyte fraction, lymphocyte infiltration, and macrophage fractions) was decreased in Late tumors compared with Survivors. Utilizing the CIBERSORT algorithm, we showed anti-cancer lymphocytes, memory CD4+ T cells and γδT cells, were significantly lower, and pro-cancerous regulatory T cells were significantly higher in Late tumors compared with Survivors. In distant recurrence analysis, we found that anti-cancer M1 macrophages were higher in Early and Mid compared to Survivors. Pro-cancerous regulatory T cells were significantly higher in Mid compared to Survivors. Interestingly, in local recurrence analysis, there was no statistically significant difference between timing of cancer recurrence and Survivors. Cytolytic activity score that assesses immune cell cytolytic activity was significantly lower in Late compared with Survivors, which suggest it to be a prognostic factor for Late recurrence. Conclusions: We demonstrated that host defense immunity, pro-cancerous immune cells, and immune cell cytolytic activity in primary breast tumors affect the timing and type of breast cancer recurrence. Citation Format: Takashi Takeshita, Li Yan, Kazuaki Takabe, Hiroko Yamashita. Pro-cancerous immune profile in primary breast tumors affects the timing and type of breast cancer recurrence [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 1607.

Abstract 4537: SBT6050, a HER2-directed TLR8 ImmunoTAC™therapeutic, is a potent human myeloid cell agonist that provides opportunity for single agent clinical activity

Abstract SBT6050, a novel therapeutic comprised of a potent toll-like receptor (TLR) 8 agonist conjugated to a HER2-directed monoclonal antibody that binds an epitope distinct from trastuzumab, is designed for systemic delivery and tumor-localized activation of human myeloid cells in the presence of moderate and high HER2-expressing tumor cells. Many solid tumors, including those expressing HER2, are refractory to immunotherapy due to immune-suppressive mechanisms, loss of HLA, low neoantigen availability, and/or minimal T cell infiltrates. These tumors frequently contain abundant populations of tumor-associated myeloid cells. Activation of these cells through TLR agonism has emerged as a promising approach in overcoming resistance mechanisms to current cancer immunotherapies. Unlike other endosomal TLRs such as TLR7 and TLR9, TLR8 is highly expressed in human myeloid cells known to be prevalent in human tumors such as conventional DCs and macrophages. TLR8's restricted myeloid cell expression removes the risk of inducing T cell death or tumor cell proliferation as described for other innate immune activators such as STING. Agonism of TLR8 in human myeloid cells activates a broad spectrum of anti-tumor immune mechanisms, including proinflammatory cytokine production, repolarization of suppressive myeloid cells and the priming of CTL responses. These functions cannot be replicated by a potent TLR7 agonist or with clinical agents such as resiquimod that agonize TLR7 and only weakly engage TLR8. Here we present data demonstrating the superiority of SBT6050 at activating human myeloid cells compared to HER2 antibody conjugates that use either a selective TLR7 agonist or resiquimod. In vitro studies with human immune cells show that SBT6050 potently induces multiple anti-tumor immune activities, including proinflammatory cytokine and chemokine production, inflammasome activation, direct activation of DCs and indirect T and NK cell cytolytic activity. Our data indicate the favorable profile of SBT6050 is likely due to efficient engagement of TLR8 in conjugate form and TLR8's unique expression profile, and not due to differences in the potency of the small molecule payloads. Using an SBT6050 mouse surrogate we show in vivo evidence for intratumoral TLR agonist activation of several myeloid driven anti-tumor pathways leading to curative single agent efficacy in both a T cell-excluded syngeneic tumor model and a xenograft model lacking T, B and NK cells. Collectively, these data demonstrate that SBT6050 displays an attractive functional profile unachievable with agonist-based antibody conjugates directed against other innate immune receptors. These data also highlight the potential for SBT6050 to be clinically active as a monotherapy. SBT6050 is projected to enter the clinic in 2020 for patients with moderate or high HER2-expressing tumors. Citation Format: Michael R. Comeau, Ty Brender, Monica Childs, Jamie Brevik, Damion Winship, Heather Metz, Jenny Chang, Jeffrey Adamo, Ben Setter, Hengyu Xu, Li-Qun Fan, Brenda Stevens, Sean W. Smith, Phil Tan, Robert DuBose, Yvette Latchman, Peter Baum, Valerie Odegard. SBT6050, a HER2-directed TLR8 ImmunoTAC™therapeutic, is a potent human myeloid cell agonist that provides opportunity for single agent clinical activity [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 4537.

Abstract 7: Gross thymic extract inhibits tumor growth by modulating the immune response in mice bearing Ehrlich ascites carcinoma cells

Abstract Purpose. Gross thymic extract (GTE) obtained from pathogen-free pigs, a product also known as Thymax has previously been shown to boost various parameters of the immune system in aged mice, as well as activating human dentritic cells and the DC-directed T-cell response in vitro. The current study examines the immunomodulatory and chemopreventive effects of GTE in mice inoculated with Ehrlich ascites carcinoma (EAC) cells. Materials and Methods. Female Swiss albino mice were inoculated with EAC cells in the thigh to develop solid tumor. Oral treatment with GTE (5.45mg/kg body weight) was started either 14 days prior to tumor cell inoculation or 9 days post inoculation for mice bearing palpable tumors. Body weight and tumor growth were monitored until day 30 post-inoculation. The percentage of infiltrated CD4+, and CD8+T cells, natural killer (NK) cell activity and TNF-α expression in tumor cells were analyzed by flow cytometry and RT-PCR. Tumor histopathology was also examined, and blood lymphocytes and safety parameters were evaluated. GTE (Thymax) was kindly provided by YS Nature Company, Tokyo, Japan. Results. Treatment with GTE prior tumor cell inoculation showed tumor incidence in 61.1 % of the animals. Pre-treatment and post-treatment with GTE, markedly delayed tumor development and resulted in tumor weight reduction by -76.7% and -48.7% of untreated control, respectively. GTE, increased blood lymphocytes count by 60% and 53%, elevated the percentage of infiltrating CD4+ cells by144%, 94% and CD8+ T cells by 146%, 53%, for pre- and post-treatment, respectively. Oral intake of GTE, profoundly augmented NK cell cytolytic activity by 5.8 and 3.1 fold and TNF-α expression by 5.5 and 3.2 fold relative to the untreated control for pre-and post-treatment, respectively. Mice treated with GTE maintained normal body, spleen, liver and kidney weights and normal values for liver function enzymes including Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) and Gamma-glutamyl transferase (GGT). Histopathological studies showed tumor tissue obtained from GTE -treated groups had a marked decrease in tumor cell density with increased vacuolation of their cytoplasm, marked increase of apoptotic cells and expansion of the degenerative areas. Conclusion: GTE is a safe, non-toxic agent, that exhibits a chemopreventive effect in mice bearing solid Ehrlich carcinoma via its ability to induce potent immunomodulation. This may suggest that GTE could be used for the treatment of solid tumors in humans. Citation Format: Azza I. Othman, Nariman K. Badr El-Din, Maggie E. Amer, Mamdooh Ghoneum. Gross thymic extract inhibits tumor growth by modulating the immune response in mice bearing Ehrlich ascites carcinoma cells [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 7.

Characterization of Neoantigen Load Subgroups in Gynecologic and Breast Cancers.

ObjectiveAlthough gynecologic and breast (Pan-Gyn) cancers share a variety of similar characteristics, their response to immunotherapy is different. Immune checkpoint inhibitor therapy is not effective in all patients, while neoantigen load (NAL) may be a predictive biomarker. However, the selection of a NAL cutoff point and its predictive effect remain to be elucidated.MethodsWe divided 812 Pan-Gyn cancer samples from The Cancer Genome Atlas into three groups based on 60 and 80% of their load percentile. We then correlated the identified NAL subgroups with gene expression, somatic mutation, DNA methylation, and clinicopathological information. We also characterized each subgroup by distinct immune cell enrichment, PD-1 signaling, and cytolytic activity. Finally, we predicted the response of each subgroup to chemotherapy and immunotherapy.ResultsAcross Pan-Gyn cancers, we identified three distinct NAL subgroups. These subgroups showed differences in biological function, genetic information, clinical variables, and immune infiltration. Eighty percent was identified as a meaningful cutoff point for NAL. In all patients, a higher NAL (top 20%) was associated with better overall survival as well as high immune infiltration and low intra-tumor heterogeneity. Furthermore, an interesting lncRNA named AC092580.4 was found, which was associated with two significantly different immune genes (CXCL9 and CXCL13).ConclusionsOur novel findings provide further insights into the NAL of Pan-Gyn cancers and may open up novel opportunities for their exploitation toward personalized treatment with immunotherapy.

Polyglutamine protein in tumor cells sensitizes tumor cells to natural killer cell cytolysis

Patients with Huntington’s diseases display reduced tumor incidence mediated by unclear mechanisms. Besides, the effects of characteristic overexpression of 97 polyglutamine protein (polyQ protein) on tumor surveillance by the host immune system have not been investigated. NK cells are cytotoxic innate lymphocytes that sense and kill stressed and transformed cells through recognition of abnormal molecular patterns. Here, we found that polyQ protein induced the accumulation of misfolded proteins in tumor cells and sensitized these tumor cells to NK cell cytolysis in vitro. Transcriptome analysis showed that polyQ protein overexpression caused an abnormal transcriptome changes in tumor cells, which might predispose these tumor cells to death upon NK cell cytolysis. However, on the other hand, polyQ protein in NK cells compromised NK cell cytolytic activity through forcing the accumulation of misfolded proteins. Furthermore, polyQ overexpression enriched oxidative phosphorylation related gene set in NK cells, which might lead to an exhaustion-like status of NK cells with reduced cytolytic activity. Therefore, our study shows that polyQ protein overexpression in tumors alone, but not in both tumor cells and NK cells, might result in increased tumor rejection by NK cells, revealing a dual role of polyQ protein on tumor surveillance by the immune system.

SBT6050, a HER2-directed TLR8 therapeutic, as a systemically administered, tumor-targeted human myeloid cell agonist.

3110 Background: Solid tumors are replete with myeloid cells which, when activated, drive potent anti-tumor responses. Clinical development of systemically administered myeloid cell agonists, however, has been hindered by acute toxicities due to peripheral activation of the targeted cell types. Intratumoral administration, the route of delivery typically used for innate immune/myeloid cell agonists, is limited by tumor accessibility and a dependence on abscopal responses. A systemically delivered myeloid cell agonist with tumor-localized activity has the potential to overcome challenges encountered with other innate immune/myeloid cell agonists in clinical development. Methods: SBT6050 is a novel therapeutic comprised of a potent toll-like receptor (TLR) 8 agonist payload conjugated to a HER2-directed monoclonal antibody. Delivery of the payload into the endosome of human myeloid cells, where TLR8 resides, requires the co-engagement of HER2 on tumor cells and Fc gamma receptor on human myeloid cells. Thus, SBT6050 is designed for systemic delivery and tumor-targeted activation of human myeloid cells. Results: Studies with human immune cells show that SBT6050 potently induces, in a HER2-dependent manner, multiple anti-tumor immune activities due to its direct activation of myeloid cells and the subsequent induction of T and NK cell cytolytic activity. SBT6050 is designed to activate human myeloid cells only in the presence of HER2-positive tumor cells with moderate (2+ by IHC) or high (3+ by IHC) expression levels. Tumor-localized activity has been demonstrated in mouse models using a SBT6050 mouse surrogate. Systemic delivery results in robust single agent efficacy in multiple mouse tumor models, even those engineered to lack T cells, without accompanying peripheral cytokine production. Trastuzumab and SBT6050 bind to distinct epitopes on HER2 and enhanced activity is observed when the two agents are combined. Conclusions: SBT6050 is a systemically administered, tumor-targeted myeloid cell agonist that demonstrates single agent efficacy in multiple mouse tumor models without peripheral cytokine production. A first-in-human study with SBT6050 is expected to begin this year for patients with HER2-expressing solid tumors.

Characterization of novel dual tandem CD19/BCMA chimeric antigen receptor T cells to potentially treat multiple myeloma

BackgroundTreatment with chimeric antigen receptor (CAR)-engineered T cells directed against the B-cell maturation antigen (BCMA) promoted transient recovery from multiple myeloma (MM). However, the absence of this antigen on immature plasma cells may limit the efficacy of this modality and facilitate relapse. The purpose of this study is to characterize a novel CAR that includes both a single-chain variable fragment (scFv)-BCMA and an scFv-CD19 in tandem orientation (tan-CAR) in an attempt to target both BCMA and CD19 expression on MM cells.MethodThe scFv sequences from the anti-CD19 antibody FMC63 and the anti-BCMA antibody C11D5.3 were ligated in tandem with transmembrane and T-cell signaling domains to generate the tan-CAR construct. Specificity and efficacy of activated tan-CAR T cells were analyzed using in vitro proliferation, cytokine release, and cytolysis assays. We also evaluated the in vivo efficacy with a xenograft mouse model that included target tumor cells that expressed CD19 or BCMA and compared the results to those obtained with conventional CAR T cells.ResultsThe in vitro studies revealed specific activation of tan-CAR T cells by K562 cells that overexpressed CD19 and/or BCMA. Cell proliferation, cytokine release, and cytolytic activity were all comparable to the responses of single scFv CAR T cells. Importantly, in vivo studies of tan-CAR T cells revealed specific inhibition of tumor growth in the mouse xenograft model that included cells expressing both CD19 and BCMA. Systemic administration of tan-CAR T cells resulted in complete tumor remission, in contrast to the reduced efficacies of BCMA-CAR T and CD19-CAR T alone in this setting.ConclusionWe report the successful design and execution of novel tan-CAR T cells that promote significant anti-tumor efficacy against both CD19 and BCMA antigen-positive tumor cells in vitro and in vivo. The data from this study reveal a novel strategy that may help to reduce the rate of relapse in the treatment with single scFv-CAR T cells.

Inducible MyD88/CD40 synergizes with IL-15 to enhance antitumor efficacy of CAR-NK cells

Natural killer (NK) cells expressing chimeric antigen receptors (CARs) are a promising anticancer immunotherapy, leveraging both innate NK cell antitumor activity and target-specific cytotoxicity. Inducible MyD88/CD40 (iMC) is a potent, rimiducid-regulated protein switch that has been deployed previously as a T-cell activator to enhance proliferation and persistence of CAR-modified T cells. In this study, iMC was extended to CAR-NK cells to enhance their growth and augment cytotoxicity against tumor cells. iMC-activated NK cells substantially increased cytokine and chemokine secretion and displayed higher levels of perforin and granzyme B degranulation. In addition, iMC activation could be coupled with ectopic interleukin-15 (IL-15) to further enhance NK cell proliferation. When coexpressed with a target-specific CAR (CD123 or BCMA), this IL-15/iMC system showed further augmented antitumor activity through enhanced CAR-NK cell expansion and cytolytic activity. To protect against potential toxicity from engineered NK cells, an orthogonal rapamycin-regulated Caspase-9 (iRC9) was included in a 4-gene, dual-switch platform. After infusion of dual-switch NK cells, pharmacologic iRC9 dimerization led to rapid elimination of a majority of expanded transduced NK cells. Thus, CAR-NK cells utilizing dual molecular switches provide an innovative and effective approach to cancer immunotherapy with controlled specificity, efficacy, and safety.

Posttransplant cyclophosphamide as a platform for immunotherapy after allogeneic stem cell transplantation for multiple myeloma

Abstract Allogeneic stem cell transplantation (alloSCT) is a highly effective, curative therapy for leukemia yet does not provide a survival benefit above autologous SCT in patients with multiple myeloma (MM). To explore this, we developed preclinical models of SCT using C57Bl/6 recipient mice and either C57Bl/6 (ASCT) or C3H.SW (alloSCT) donor grafts. Importantly, these models recapitulated the clinical setting whereby alloSCT provided superior outcomes, compared to ASCT, in recipients bearing MLL-AF9-driven acute myeloid leukemia (AML) but not in those bearing Vk*MYC-MM. Interestingly, we found that MM-specific, T cell-mediated immunity was generated after ASCT, which failed due to MM-induced T cell exhaustion. MM relapse after ASCT could be prevented by TIGIT or PD-1 targeted immune checkpoint inhibition or via depletion of suppressive CSF1-R+ myeloid cells. Conversely, after alloSCT, T cell exhaustion was driven principally by alloantigen and CD8 T cells expressed high levels of PD-1, TIGIT and TIM-3. Furthermore, Vk*MYC myeloma exploited this alloantigen-driven T cell exhaustion via expression of high levels of PD-L1 and CD155 (the cognate ligands for PD-1 and TIGIT), which were expressed minimally on MLL-AF9 AML. To exploit alloSCT in MM, we used post-transplant cyclophosphamide (PT-Cy) to delete high affinity alloreactive T cells that generate the exhausted donor T cell pool. Subsequent administration of CD137 agonists enhanced T cell activation and cytolytic activity within bone marrow, without exacerbating GVHD. Thus, PT-Cy provides a platform for optimizing immunotherapy after alloSCT.

Combining a novel dual RAF/MEK inhibitor with immunomodulation to promote an anti-tumor response

Abstract The therapeutic scope of MEK inhibitors (MEKis) is currently limited to use in BRAF mutant melanoma. Therefore, we aim to develop new strategies to extend their usage to MEKi resistant RAS mutant cancers, which represent an unmet clinical need. A strategy we investigated is to balance the positive and negative immunomodulatory effects of MEKis for optimal combination with immunotherapy. In Ras mutant murine lung cancers, CH5126766 (CKI27) is novel due to its ability to inhibit both RAF and MEK, preventing the rebound of ERK that normally results from the relief of negative feedback in the MAPK pathway. We observed that CKI27 increased MHC expression on tumor cells and T cell mediated killing. Yet, CKI27 also decreased T cell proliferation, activation, and cytolytic activity. Implementing a break for T cells to recover with intermittent dosing of CKI27 partially relieved these inhibitory effects. Further combination with co-stimulatory agonist antibodies targeting OX40 and GITR completely alleviated these T cell toxicities and increased combination efficacy with checkpoint blockade antibody anti-CTLA-4. Understanding the immunomodulatory effects of combining CKI27 with immunotherapy will elucidate the mechanism behind their increased efficacy. This will allow us to make more informed decisions in dosing regimens, overcoming resistance, and generating long-term immune responses in current and future clinical trials treating patients with RAS mutant cancers.

Zebrafish pten Genes Play Relevant but Distinct Roles in Antiviral Immunity.

The PTEN (phosphatase and TENsin homolog on chromosome 10) gene encodes a bifunctional phosphatase that acts as a tumor suppressor. However, PTEN has been implicated in different immune processes, including autophagy, inflammation, regulation of natural killer (NK) cell cytolytic activity and type I interferon responses. Unlike mammals, zebrafish possess two pten genes (ptena and ptenb). This study explores the involvement of both zebrafish pten genes in antiviral defense. Although ptena−/− and ptenb−/− larvae were more susceptible to Spring viremia of carp virus (SVCV), the viral replication rate was lower in the mutant larvae than in the wild-type larvae. We observed that both mutant lines showed alterations in the transcription of numerous genes, including those related to the type I interferon (IFN) system, cytolytic activity, autophagy and inflammation, and some of these genes were regulated in opposite ways depending on which pten gene was mutated. Even though the lower replication rate of SVCV could be associated with impaired autophagy in the mutant lines, the higher mortality observed in the ptena−/− and ptenb−/− larvae does not seem to be associated with an uncontrolled inflammatory response.

Lymphovascular invasion in breast cancer is associated with gene expression signatures of cell proliferation but not lymphangiogenesis or immune response.

While the prognostic relevance of lymphovascular invasion (LVI) in breast cancer is well known, its molecular biology is poorly understood. We hypothesized that pathologically determined LVI reflects molecular features of tumors and can be discerned from their genomic and transcriptomic profiles. LVI status and Nottingham histological scores of primary breast tumors of The Cancer Genome Atlas (TCGA) project were assessed from pathology reports; other clinical and molecular data were obtained from TCGA data portals and publications. Two independent datasets (GSE5460 and GSE7849) were combined and used for validation. LVI status was determinable for 639 and 196 cases of the TCGA and validation cohorts, among whom LVI incidence was 37.8% and 37.2%, respectively. LVI was associated with high tumor Ki67 expression, advanced pathologic stage, and high Nottingham scores. LVI-positive cases had worse overall and progression-free survival regardless of cancer subtype. Surprisingly, in both cohorts, LVI was not associated with lymphangiogenesis or lymphatic vessel density as estimated from tumor expression of lymphatic endothelium-associated genes. LVI-positive tumors had higher genome copy number aberrations, aneuploidy, and homologous recombination defects, but not single-nucleotide variations or intra-tumor genome heterogeneity. Tumor immune cell composition and cytolytic activity was not associated with LVI status. On the other hand, expression of cell proliferation-related genes was significantly increased in LVI-positive tumors. Our study suggests that breast cancer with LVI is a highly proliferative cancer, and it does not correlate with gene expression markers for lymphangiogenesis or immune response.