Immune-mediated Killing Research Articles

Pharmacologic Inhibition of FGFR Modulates the Metastatic Immune Microenvironment and Promotes Response to Immune Checkpoint Blockade.

The effectiveness of immunotherapy as a treatment for metastatic breast cancer is limited due to low numbers of infiltrating lymphocytes in metastatic lesions. Herein, we demonstrated that adjuvant therapy using FIIN4, a covalent inhibitor of fibroblast growth factor receptor (FGFR), dramatically delayed the growth of pulmonary metastases in syngeneic models of metastatic breast cancer. In addition, we demonstrated in a syngeneic model of systemic tumor dormancy that targeting of FGFR enhanced the immunogenicity of the pulmonary tumor microenvironment through increased infiltration of CD8+ lymphocytes and reduced presence of myeloid suppressor cells. Similar impacts on immune cell infiltration were observed upon genetic depletion of FGFR1 in tumor cells, which suggested a direct influence of FGFR signaling on lymphocyte trafficking. Suppression of CD8+ lymphocyte infiltration was consistent with FGFR-mediated inhibition of the T-cell chemoattractant CXCL16. Initial attempts to concomitantly administer FIIN4 with immune checkpoint blockade failed due to inhibition of immune-mediated tumor cell killing via blockade of T-cell receptor signaling by FIIN4. However, this was overcome by using a sequential dosing protocol that consisted of FIIN4 treatment followed by anti-PD-L1. These data illustrate the complexities of combining kinase inhibitors with immunotherapy and provide support for further assessment of FGFR targeting as an approach to enhance antitumor immunity and improve immunotherapy response rates in patients with metastatic breast cancer.

Sero-prevalence and risk factors for hepatitis B virus infection among the consumers of the alcoholic beverage, cheka in Konso zone, southwestern Ethiopia

IntroductionHepatitis B virus (HBV) is a hepatotropic and partially double stranded deoxyribonucleic acid (DNA) virus that causes the immune-mediated killing of infected liver cells. It is a public health conundrum due to its considerable morbidity and mortality. Cheka is a cereal and vegetable-based fermented beverage that is consumed as a key dietary component in the southwestern parts of Ethiopia. This study was aimed to assess the seroprevalence of hepatitis B surface antigen (HBsAg) and associated risk factors among cheka consumers in the Konso community, southwest of Ethiopia. MethodsA community-based cross-sectional study was conducted in a total of 633 cheka consumers between October 01, 2018, and May 31, 2019. The socio-demographic characteristics and associated factors were collected using a pre-tested semi-structured questionnaire. HBV infection was screened using immune-chromatographic strip tests and positive results were further confirmed with ELISA. ResultsThe prevalence of HBV was found to be 18.2% (95% CI, 15.1–21.5). Gender (male) (AOR = 1.757:95% CI, 1.009–3.059), age (35–44 yrs), (AOR = 4.123:95% CI, 1.718–9.892), nature of the job (daily laborer) (AOR = 4.551:95% CI, 1.115–18.57), awareness about the transmission of the disease (AOR = 2.056:95% CI, 1.074–3.934) and previous history of contacts with patients having liver diseases (AOR = 2.496:95% CI, 1.187–5.248) were identified as factors which are being significantly associated with the HBV infection. ConclusionsVery high endemic levels of HBV infection are identified in the study. Male participants of age between 35 and 44 years, who are daily laborers and are aware of HBV transmission, with a previous history of contacts with patients having liver diseases were even at higher risk of HBV infection. The need for more research to find the links between cheka consumption and other mechanisms of HBV cross-infection in these communities is indicated.

Preclinical and Translational Data Support Development of Iberdomide in Combination with CD38- and SLAMF7-Directed Monoclonal Antibodies: Evidence for Rational Combinations

Preclinical and Translational Data Support Development of Iberdomide in Combination with CD38- and SLAMF7-Directed Monoclonal Antibodies: Evidence for Rational Combinations

SBRT-Partial Tumor Irradiation for The Treatment of Unresectable Bulky NSCLC

to report on the improved therapeutic ratio by partial tumor irradiation exploiting the bystander (BE) and abscopal effects (AE) for the treatment of bulky NSCLC. Unlike conventional (whole tumor + CTV + PTV) RT, in order to generate predominant immune-mediated tumor cell killing by radiation, we developed SBRT-PArtial Tumor irradiation targeting HYpoxic segment (SBRT-PATHY) while sparing and preserving the functionality of peritumoral immune microenvironment (PIM) and the regional lymphocytes necessary to mediate BE/AE. Our preclinical studies showed that the hypoxic tumor sub-volume if selectively irradiated was the “most-abscopal one” with highest inductive abscopal potential compared with normoxic segment. Our hypothesis is that SBRT-PATHY can improve the therapeutic ratio of unresectable bulky NSCLC by exploiting BE and AE. The primary endpoint was bulky tumor control rate. The secondary endpoints included AE rate, survival and toxicity. 20 patients with inoperable stage IIIB/IV bulky NSCLC considered unsuitable for conventional RT-chemotherapy due to comorbidities or tumor site and size were included. Combination of CT and 18F-FDG-PET was used per institutional protocol to define the hypovascularized (contrast-hypo-enhanced) and hypometabolic (SUV max < 3) tumor segment which was irradiated with 10Gy × 3 to the 70%-isodose. PIM was contoured as 1cm large peritumoral tissue that was spared from radiation (applied dose constraints: Dmin<1Gy/fraction, Dmax<5Gy/fraction, Dmean<3Gy/fraction). Immunohistochemistry and gene expression analysis were performed on the available tissue samples of 3 patients submitted to surgery following SBRT-PATHY in order to extract responding partially irradiated and unirradiated-abscopal tumor lesions. Median follow up was 12.9 months (range: 4-27). Mean bulky diameter was 8.4 cm (range: 6-14.5). On an average, the irradiated volume corresponded to 32% of the total tumor volume. The bulky tumor control rate was 95% (average bulky shrinkage 71%). AE was observed in 46% of patients. Overall, cancer-specific and progression-free survival were 55%, 75% and 65%, respectively. Multi-variate analysis for cancer specific survival was significant for SBRT-PATHY independent of age, sex, performance status, histology, stage, bulky site and volume (p < 0.001). 6 patients experienced fatigue grade 1. An effective antitumor immune response was activated in radiation-spared PIM leading to cell death of the partially irradiated tumors. CD20+ B-lymphocytes, CD3+/CD8+ T-lymphocytes densely infiltrated PIM and the border of the partially irradiated tumors. Apoptotic abscopal tumor sites showed a strong expression of the cell death-inducing cytokines, TGF-beta and FAS ligand and to a lesser degree of TRAIL and TNF-alpha. SBRT-PATHY for unresectable bulky NSCLC showed inspiring results that may potentially enhance the radiotherapy therapeutic ratio. A larger prospective trial is ongoing.

Healthy Donors Harbor Memory T Cell Responses to RAS Neo-Antigens.

Simple SummaryMemory T cells from healthy individuals respond to stimulation with peptides derived from cancer-specific RAS mutations, thus suggesting that healthy individuals have been challenged with RAS mutations earlier in life.The RAS mutations are the most frequently occurring somatic mutations in humans, and several studies have established that T cells from patients with RAS-mutant cancer recognize and kill RAS-mutant cells. Enhancing the T cell response via therapeutic cancer vaccination against mutant RAS results in a clinical benefit to patients; thus, T cells specific to RAS mutations are effective at battling cancer. As the theory of cancer immuno-editing indicates that healthy donors may clear malignantly transformed cells via immune-mediated killing, and since T cells have been shown to recognize RAS-mutant cancer cells, we investigated whether healthy donors harbor T-cell responses specific to mutant RAS. We identified strong and frequent responses against several epitopes derived from the RAS codon 12 and codon 13 mutations. Some healthy donors demonstrated a response to several mutant epitopes, and some, but not all, exhibited cross-reactivity to the wild-type RAS epitope. In addition, several T cell responses were identified against mutant RAS epitopes in healthy donors directly ex vivo. Clones against mutant RAS epitopes were established from healthy donors, and several of these clones did not cross-react with the wild-type epitope. Finally, CD45RO+ memory T cells from healthy donors demonstrated a strong response to several mutant RAS epitopes. Taken together, these data suggest that the immune system in healthy donors spontaneously clears malignantly transformed RAS-mutant cells, and the immune system consequently generates T-cell memory against the mutations.

Artificial selection for host resistance to tumour growth and subsequent cancer cell adaptations: an evolutionary arms race

BackgroundCancer progression is governed by evolutionary dynamics in both the tumour population and its host. Since cancers die with the host, each new population of cancer cells must reinvent strategies to overcome the host’s heritable defences. In contrast, host species evolve defence strategies over generations if tumour development limits procreation.MethodsWe investigate this “evolutionary arms race” through intentional breeding of immunodeficient SCID and immunocompetent Black/6 mice to evolve increased tumour suppression. Over 10 generations, we injected Lewis lung mouse carcinoma cells [LL/2-Luc-M38] and selectively bred the two individuals with the slowest tumour growth at day 11. Their male progeny were hosts in the subsequent round.ResultsThe evolved SCID mice suppressed tumour growth through biomechanical restriction from increased mesenchymal proliferation, and the evolved Black/6 mice suppressed tumour growth by increasing immune-mediated killing of cancer cells. However, transcriptomic changes of multicellular tissue organisation and function genes allowed LL/2-Luc-M38 cells to adapt through increased matrix remodelling in SCID mice, and reduced angiogenesis, increased energy utilisation and accelerated proliferation in Black/6 mice.ConclusionHost species can rapidly evolve both immunologic and non-immunologic tumour defences. However, cancer cell plasticity allows effective phenotypic and population-based counter strategies.

109 Poster - The non-peptidomimetic cIAP1/2 and XIAP antagonist ASTX660 promotes an anti-tumour immune response in T-cell lymphoma

Background: ASTX660 is a potent, non-peptidomimetic antagonist of the cellular and X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), which is currently being tested in a first in human phase I-II study in patients with advanced solid tumours and lymphomas (NCT02503423). IAP antagonists have been reported to exhibit broad immuno-modulatory effects on both the innate and adaptive immune systems. We have investigated the profile of ASTX660 in preclinical T cell lymphoma models and evaluated ASTX660’s ability to enhance immune mediated killing of T cell lymphoma cells, both in vitro and in vivo.

Antibody and antibody fragments for cancer immunotherapy

Antibody has become the most rapidly expanding class of pharmaceuticals for treating a wide variety of human diseases including cancers. Especially, with the fast development of cancer immunotherapy, antibody drugs have become the most promising therapeutic for curing cancers. Immune-mediated cell killing by antibodies including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) as well as regulation of T cell function through immune checkpoint blockade. Due to the absence of Fc fragment, antibody fragments including single-chain variable fragments (scFvs) and single-domain antibodies (sdAds) are mainly applied in chimeric antigen receptors (CAR) T cell therapy for redirecting T cells to tumors and T cell activation by immune checkpoint blockade. In this review, the cancer immunity is first discussed. Then the principal mechanisms of antibody-based immunotherapy will be reviewed. Next, the antibody and antibody fragments applied for cancer immunotherapy will be summarized. Bispecific and multispecific antibodies and a combination of cancer immunotherapy with other tumor treatments will also be mentioned. Finally, an outlook and perspective of antibody-based cancer immunotherapy will be given. This review would provide a comprehensive guidance for the researchers who are interested in and intended to involve in the antibodies- or antibody fragments-based tumor immunity.

Abstract 5542: ASTX660, a non-peptidomimetic antagonist of cIAP1/2 and XIAP, enhances immune mediated tumor killing and induction of immunogenic cell death

Abstract Introduction: ASTX660 is a potent, non-peptidomimetic antagonist of the cellular and X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), which is currently being tested in a first in human phase I-II study in patients with advanced solid tumors and lymphomas (NCT02503423). IAP antagonists enhance tumor necrosis factor receptor superfamily mediated cell death and enhance anti-tumor immune responses. Herein, we describe the profile of ASTX660 in a range of preclinical tumor models and evaluate ASTX660's ability to enhance immune mediated killing of tumor cells, both in vitro and in vivo. Methods: ASTX660 was tested in a panel of human and mouse tumor cell lines, assessing proliferation, apoptosis and immunogenic cell death (ICD). ASTX660 was tested alone or with recombinant Death Receptor ligands. Additionally, we used a novel co-culture system of tumor cell lines with anti-CD3 activated human peripheral blood mononuclear cells (PBMC) to assess ASTX660 effects on immune mediated cell killing. Target engagement along with induction of apoptosis and ICD markers were analysed by Western blotting and flow cytometry. Murine tumor models in immunocompetent and immunocompromised mice were utilised to evaluate the efficacy of ASTX660 in the presence or absence of an effective immune response. Results: ASTX660 antagonised IAPs in cell lines, as indicated by a decrease in cIAP1 protein levels and disruption of the XIAP:SMAC protein complex. In murine T cell lymphoma cell lines ASTX660 treatment was associated with an increase in apoptosis and ICD. In immunocompetent mice, administration of ASTX660 delivered a complete regression of BW5147 tumor growth which was not seen in mice deficient in T and B cells. To confirm human relevance, ASTX660 also enhanced anti-CD3 stimulated PBMC-dependent killing of tumour cell lines. Conclusion: The combination of the direct effects of ASTX660 on tumor cells and ASTX660 dependent effects on immune responses adds to the description of ASTX660's mode of action and our ongoing understanding of the clinical efficacy in T cell Lymphoma(1). Reference: A Hollebecque et al. 2019 AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics. Citation Format: Nicola Ferrari, George Ward, Joanne Munck, Simone Jueliger, Matthew Davis, Christina Gewinner, Roberta Ferraldeschi, John Lyons, Martin John Sims. ASTX660, a non-peptidomimetic antagonist of cIAP1/2 and XIAP, enhances immune mediated tumor killing and induction of immunogenic cell death [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 5542.

Abstract CT014: IMbassador250: A phase III trial comparing atezolizumab with enzalutamide vs enzalutamide alone in patients with metastatic castration-resistant prostate cancer (mCRPC)

Abstract Background: Current treatments for mCRPC include the androgen receptor antagonist enzalutamide (enza). Enza may enhance IFNγ signaling and sensitize tumor cells to immune-mediated cell killing, making it a candidate for combinations with PD-L1/PD-1 inhibitors. Responses have been observed in patients (pts) with mCRPC receiving a PD-L1/PD-1 inhibitor with or without enza. Therefore, atezolizumab (atezo), a humanized anti-PD-L1 monoclonal antibody that inhibits the interaction between PD-L1 and its receptors, is being examined in combination with enza for pts with mCRPC. Methods: IMbassador250 was a Phase III, randomized clinical trial (NCT03016312) evaluating the safety and efficacy of atezo + enza vs enza alone in pts with mCRPC who had progressed on abiraterone and docetaxel, or were not candidates for a taxane regimen (androgen deprivation therapy was not given). Eligible pts had mCRPC or locally advanced, incurable CRPC, ECOG PS 0/1 and no prior immunotherapy or enza/androgen receptor antagonist treatment. After a safety run-in, pts were randomized 1:1 to receive atezo 1200 mg every 3 weeks + enza 160 mg once daily or enza 160 mg once daily only. Stratification factors were prior taxane treatment, presence of liver metastases, level of circulating LDH and pain. The primary endpoint was OS; secondary endpoints included PSA response rate, radiographic PFS, ORR and safety. Tumor tissue and disease progression will be used to assess the relationship between exploratory biomarkers and treatment response. Results: A total of 759 pts were randomized to atezo + enza (n = 379) or enza alone (n = 380). Baseline demographic and clinical characteristics were generally similar between groups. The primary efficacy endpoint of OS was evaluated (data cutoff for efficacy and safety, June 24, 2019), with no difference observed between groups (stratified HR, 1.12 [95% CI: 0.91, 1.37]; P = 0.28). Median OS was 15.2 months (95% CI: 14.0, 17.0) in the atezo + enza group and 16.6 months (95% CI: 14.7, 18.4) in the enza group. Subgroup analyses were consistent with the primary endpoint. Analyses of secondary and exploratory endpoints are ongoing and will be presented. Adverse events (AEs) were consistent with those seen with the treatment components. Nearly all pts had an AE (atezo + enza, 96.5%; enza alone, 91.8%). Treatment-related AEs (TRAEs) were observed in 77.8% and 51.1% of pts, respectively, with Grade 3-4 TRAEs in 28.3% and 9.6% and Grade 5 TRAEs in 1.9% and 0.3% of pts. Conclusions: The combination of atezo + enza did not show an OS improvement vs enza alone, requiring early termination of the study. The observed safety profiles were consistent with the known safety profiles of each treatment. Analyses of secondary outcomes and exploratory endpoints, biomarker studies and treatment response may provide further insight into these findings. Citation Format: Christopher J. Sweeney, Silke Gillessen, Dana Rathkopf, Nobuaki Matsubara, Charles Drake, Karim Fizazi, Josep M. Piulats, Piotr J. Wysocki, Gary L. Buchschacher Jr, Jennifer Doss, Sanjeev Mariathasan, Edward E. Kadel, Kobe Yuen, Asim Datye, Grozdana Rasuo, Darren Tayama, Patrick Williams, Thomas Powles. IMbassador250: A phase III trial comparing atezolizumab with enzalutamide vs enzalutamide alone in patients with metastatic castration-resistant prostate cancer (mCRPC) [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 CT014.

Sindbis Virus with Anti-OX40 Overcomes the Immunosuppressive Tumor Microenvironment of Low-Immunogenic Tumors

Despite remarkable responses to cancer immunotherapy in a subset of patients, many patients remain resistant to therapies. It is now clear that elevated levels of tumor-infiltrating T cells as well as a systemic anti-tumor immune response are requirements for successful immunotherapies. However, the tumor microenvironment imposes an additional resistance mechanism to immunotherapy. We have developed a practical and improved strategy for cancer immunotherapy using an oncolytic virus and anti-OX40. This strategy takes advantage of a preexisting T cell immune repertoire in vivo, removing the need to know about present tumor antigens. We have shown in this study that the replication-deficient oncolytic Sindbis virus vector expressing interleukin-12 (IL-12) (SV.IL12) activates immune-mediated tumor killing by inducing OX40 expression on CD4 T cells, allowing the full potential of the agonistic anti-OX40 antibody. The combination of SV.IL12 with anti-OX40 markedly changes the transcriptome signature and metabolic program of T cells, driving the development of highly activated terminally differentiated effector T cells. These metabolically reprogrammed T cells demonstrate enhanced tumor infiltration capacity as well as anti-tumor activity capable of overcoming the repressive tumor microenvironment. Our findings identify SV.IL12 in combination with anti-OX40 to be a novel and potent therapeutic strategy that can cure multiple types of low-immunogenic solid tumors.

Granzyme B PET imaging of immune-mediated tumor killing as a tool for understanding immunotherapy response

BackgroundCancer immunotherapy research is expanding to include a more robust understanding of the mechanisms of treatment response and resistance. Identification of drivers of pro-tumor and anti-tumor immunity during treatment offers...

STING pathway mediated immunologic consequences of DNA damage repair gene losses in high-grade serous ovarian tumors

Abstract High grade serous ovarian carcinoma (HGSC) is the most lethal gynecologic malignancy with high rates of chemotherapy resistance and poor outcomes. Our previous studies have demonstrated the variable tumor immune microenvironment states that associate with platinum chemotherapy response. We further showed the significance of the interferon induced chemokine CXCL10 as a key mediator of tumor infiltrating immune cell recruitment. Using the ID8-Trp53−/− murine model of HGSC, we demonstrated the potential of Stimulator of Interferon Genes (STING) pathway activation in enhancing response of HGSC tumors to carboplatin chemotherapy and sensitizing them to immune checkpoint blockade. Notably, CXCL10 production via IFN1 is also governed by genes that regulate cellular DNA damage repair pathways. Evolving evidence indicates a role of BRCA1 and PTEN genes in mediating cellular IFN1 responses. Losses in the function of these genes is widely prevalent in a large proportion of HGSC tumors, where tumors with BRCA1 mutations have higher CD8+ T cell infiltration in contrast to those with loss of PTEN. We hypothesized that HGSC tumors with loss of PTEN expression can be rendered susceptible to immune mediated killing via activating the STING pathway. Tumors generated from ID8-Trp53−/−; Brca1−/− cells and those from ID8-Trp53−/−; Pten−/− cells in C57BL6 mice showed significant immunologic differences. STING agonist treatment significantly increased chemosensitivity and improved overall response in mice implanted with ID8-Trp53−/−; Pten−/− cells compared to those treated with carboplatin alone, altering immune responses. This study is foundational to guide rationalistic combinations of STING pathway activating therapies in HGSC.

Tumor RIPK1 signaling mediates intrinsic and extrinsic mechanisms of resistance to immune checkpoint blockade therapy

Abstract Immune checkpoint blockade (ICB) therapy can result in impressive clinical responses of multiple cancer types, however resistance is common. Mechanisms of resistance include both tumor intrinsic evasion of immune mediated killing and tumor extrinsic features in the immune-microenvironment, such as poor infiltration of tumor reactive T cells and accumulation of immunosuppressive cells. By examining transcriptomic features associated with response or resistance to ICB, we found the expression of Receptor Interacting Protein Kinase 1 (RIPK1), a critical regulator of inflammation and cell death, is significantly higher in ICB resistant murine tumor cells. We hypothesized that RIPK1 expression in the tumor cells may influence both intrinsic and extrinsic mechanisms regulating response to ICB. To test this hypothesis, we genetically deleted RIPK1 in murine tumor cell lines and found improved overall survival upon treatment compared to tumors expressing RIPK1. Mechanistically, deletion of RIPK1 in the tumor cells results in favorable changes in the tumor microenvironment, including an increased frequency of effector T cells, a decreased frequency of immunosuppressive myeloid cells, and changes in inflammatory cytokine secretion that can impact these changes in immune infiltrate. Further, deletion of the apoptosis effector Caspase 8 reversed RIPK1 KO tumor sensitization to ICB, suggesting that tumor sensitization to cell death also contributes to improved response to ICB upon RIPK1 deletion. In conclusion, RIPK1 is an important regulator at the intersection of tumor intrinsic and extrinsic pathways governing response to ICB, and therefore is a potential target for combinatorial therapies to improve patient response to ICB.

Abstract B04: Multimodal cancer immunotherapy combining IL-8 inhibition, adenovirus vaccine, IL-15 super agonist, and anti-PD-L1/TGFβRII agent reduces mesenchymalization and enhances anti-tumor efficacy

Abstract Tumor cells utilize the secretion of proinflammatory cytokines and chemokines and the surface expression of inhibitory ligands to promote an immune-suppressive tumor microenvironment that facilitates tumor growth and metastasis. The overexpression of IL-8, TGF-β, PD-L1, the IL-8 receptors CXCR1 and CXCR2, as well as numerous other factors by various solid and hematologic cancers are major obstacles in the promotion of a strong and effective antitumor immune response. Both IL-8 and TGF-β have been identified as key potentiators of tumor cell plasticity and the acquisition of mesenchymal features by epithelial cancer cells, i.e., mesenchymalization, which increases metastatic dissemination. Both IL-8 and TGF-β can also promote tumor angiogenesis, myeloid-derived suppressor cell (MDSC) function in the tumor microenvironment, and resistance to immune-mediated killing. Additionally, tumor cell expression of PD-L1 inhibits effective T-cell responses at the tumor site. Here we report the use of a systematic and rational multimodal cancer immunotherapy approach that combines induction of T cell potentiation through an IL-15 super agonist and adenoviral cancer vaccine with inhibition of three immunosuppressive pathways through blockade of IL-8, TGF-β, and PD-L1. Combination of the novel small-molecule inhibitor SX-682 that irreversibly binds and blocks both CXCR1 and CXCR2 with an adenoviral-based cancer vaccine, N-IL15 (previously designated ALT-803), and the anti-PD-L1/TGFβRII agent, M7824, significantly delayed tumor growth in murine models of breast and colon cancer. Mechanistic studies demonstrated that this multimodal combination therapy was able to markedly increase tumor infiltration with CD4+ and CD8+ T cells, including central memory and effector memory subsets, and to decrease infiltration with Ly6G+/Ly6C- MDSC. Studies with SX-682 as a monotherapy suggest that CXCR1/2 inhibition reduces migration rates of Ly6G+/Ly6C- MDSC (CXCR2+). Studies with models of claudin-low, mesenchymal-enriched, human triple-negative breast cancer demonstrated that CXCR1/2 inhibition was also effective at promoting a switch from a mesenchymal to an epithelial tumor phenotype, while simultaneously enhancing the tumor cells’ susceptibility to immune-mediated cytotoxicity. Our results provide rationale for the future evaluation of this multimodal therapy in the clinic. Citation Format: Lucas Horn, Duane Hamilton, Dean Maeda, John Zebala, John Lee, Shahrooz Rabizadeh, Frank Jones, Patrick Soon-Shiong, Zhen Su, Jeffrey Schlom, Claudia Palena. Multimodal cancer immunotherapy combining IL-8 inhibition, adenovirus vaccine, IL-15 super agonist, and anti-PD-L1/TGFβRII agent reduces mesenchymalization and enhances anti-tumor efficacy [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 B04.

Azithromycin Exerts Bactericidal Activity and Enhances Innate Immune Mediated Killing of MDR Achromobacter xylosoxidans.

Azithromycin (AZM), the most commonly prescribed antibiotic in the United States, is thought to have no activity against multidrug-resistant Gram-negative pathogens such as Achromobacter xylosoxidans (AX) per standard minimum inhibitory concentration testing in cation-adjusted Mueller Hinton Broth. Here we provide the first report of AZM bactericidal activity against carbapenem-resistant isolates of AX, with a multifold decrease in minimum inhibitory concentration across 12 clinical isolates when examined under physiologic testing conditions that better recapitulate the in vivo human environment. This pharmaceutical activity, evident in eukaryotic tissue culture media, is associated with enhanced AZM intracellular penetration and synergistic killing with human whole blood, serum, and neutrophils. Additionally, AZM monotherapy inhibited preformed AX biofilm growth in a dose-dependent manner together with a reduction in viable bacteria. In an illustrative case, AZM in combination with piperacillin-tazobactam exerted clear therapeutic effects in a patient with carbapenem-resistant AX mediastinitis, sternal osteomyelitis, and aortic graft infection. Our study reinforces how current antimicrobial testing practices fail to recapitulate the host environment or host-pathogen interactions and may misleadingly declare complete resistance to useful agents, adversely affecting patient outcomes. We conclude that AZM merits further exploration in the treatment of drug-resistant AX infections. Novel approaches to antimicrobial susceptibility testing that better recapitulate the host environment should be considered, especially as infections caused by multidrug-resistant Gram-negative bacterial pathogens are expanding globally with high morbidity and mortality.

Abstract A43: Exploiting off-target effects of estrogen receptor-targeting drugs to sensitize breast cancer to immune killing

Abstract 1 in 8 women will be diagnosed with breast cancer within their lifetime. The majority of women are diagnosed with estrogen receptor-positive (ER+), progesterone receptor-positive (PR+), or HER2+ breast cancers. The standard of care for ER+ breast cancer is treatment with selective estrogen receptor modulator (SERM) and selective estrogen receptor degrader (SERD) drugs. While these are highly effective for ER+ breast cancers and additional targeted drugs exist for PR+ and HER2+ cancers, there are currently no targeted therapeutic strategies for the 10-15% of women who are diagnosed with triple-negative breast cancer (TNBC). We hypothesize that estrogen receptor-targeting drugs are capable of inducing phenotypic changes to sensitize breast tumor cells to immune-mediated killing regardless of their ER status. In this study, we used in vitro killing assays and molecular characterization of ER+ and ER- cell lines to identify that SERM and SERD drugs sensitize breast cancer to killing by healthy-donor natural killer (NK) cells regardless of their ER status. While tamoxifen (SERM) and fulvestrant (SERD) had no cytotoxic or antiproliferative effect on ER- cells, pretreatment with either resulted in increased NK-mediated cell lysis. Through RNAseq analysis of tamoxifen-treated ER+ and ER- cells, we identified increased activation of both apoptotic and death receptor signaling pathways. These pathways play a key role in immunogenic modulation, indicating a potential mechanism for tamoxifen-induced tumor sensitization to immune cell lysis. It has previously been reported that in addition to binding to ER, both tamoxifen and fulvestrant bind to G protein-coupled estrogen receptor 1 (GPR30). We found that GPR30 is expressed in both ER+ and ER- breast cancer cells. Using the specific GPR30 agonist G-1, we demonstrated that targeted activation of GPR30 signaling resulted in increased NK cell killing. These data indicate that the observed ER-independent immunogenic modulation of breast cancer cells may be occurring through GPR30. Together, these findings demonstrate a novel effect of SERM and SERD drugs on the interaction between both ER+ and ER- tumor cells with the immune system. This study is the first to demonstrate the potential use of ER-targeting drugs in immunotherapy combinations in an ER-agnostic manner and may inform novel immunotherapy strategies in breast cancer. Citation Format: Benjamin Wolfson, Jeffrey Schlom, James Hodge. Exploiting off-target effects of estrogen receptor-targeting drugs to sensitize breast cancer to immune killing [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 A43.

Pre-therapeutic Evaluation of Patient-Specific Responses to Immune-Checkpoint Inhibition in Colorectal Cancer

Abstract Background Cancer immunotherapy has revolutionized treatment options for patients suffering from immunogenic tumors, among them melanoma, bladder and lung cancer. In the context of colorectal cancer (CRC), the administration of checkpoint inhibitors (CPIs) was shown to induce durable clinical responses in patients with mismatch repair deficient and microsatellite instable tumors, a though very small fraction of all CRC subtypes. To ameliorate response rates in metastatic CRC, various therapeutic strategies are currently being investigated to increase the immunogenicity of mismatch repair proficient (pMMR)/ microsatellite stable (MSS) CRC tumors and to set the ground for immunotherapy. Several conventional therapies approved for treating CRC patients are classified as immunogenic cell death inducers and may aid in priming cytotoxic T cells to the patient’s tumor. Methods At 2cureX, we aim to pre-therapeutically measure the potential responsiveness of a CRC patient to various drug therapy options to support the oncologists in identifying the best suitable treatment regimen. Our functional IndiTreat® assay system allows for testing of chemotherapeutic agents, targeted therapies and combinations thereof against micro-tumors (tumoroids), which we derive from CRC tissue or liver metastases. To broaden the applicability of the IndiTreat® test, the present study aims to adapt the assay system to functional testing of checkpoint inhibitors in the context of pMMR/MSS CRC, subsequent to standard of care therapy. Results To assess the potential efficacy of IO interventions for individual patients, we co-culture tumoroids and (in vitro expanded) autologous PBMCs and monitor immune-mediated killing of tumoroids in the presence of CPIs. Tumoroids recapitulate the highly individual disease of cancer patients and constitute a valuable platform for evaluating different aspects of immune-mediated tumor cell recognition and killing. Conclusion In vitro testing of individual responses to CPIs will be of key relevance for stratifying pre-treated pMMR/MSS CRC patients according to their likelihood to benefit from IO therapy and holds the potential to make these powerful drugs available to more patients suffering from CRC. Legal entity responsible for the study The authors. Funding 2cureX, BMBF KMU-innovativ. Disclosure T. Sturmheit: Full / Part-time employment: 2cureX. T. Sutus Temovski: Full / Part-time employment: 2cureX. J. Thastrup: Full / Part-time employment: 2cureX. W. Fiedler: Advisory / Consultancy, Research grant / Funding (self), Licensing / Royalties: Amgen; Advisory / Consultancy: ARIAD/ Incyte; Advisory / Consultancy: Novartis; Advisory / Consultancy: Pfizer; Advisory / Consultancy: Jazz Pharmaceuticals; Research grant / Funding (self): Pfizer. J. Wellbrock: Licensing / Royalties: Amgen. J. Kupper: Full / Part-time employment: 2cureX. A. Block: Research grant / Funding (self): 2cureX. All other authors have declared no conflicts of interest.

Abstract C056: MIV-818 stimulates an antitumor immune response in vitro and enhances the effects of pembrolizumab

Abstract Purpose: Investigation of immune-modulating activity of MIV-818. Background: MIV-818, a phosphoramidate prodrug of troxacitabine was designed to target the active metabolite troxacitabine-triphosphate to the liver while minimizing systemic exposure. MIV-818 is being developed for the treatment of cancer in the liver, and is currently being evaluated in a phase 1/2 study. Preclinical studies have demonstrated that some DNA damaging agents may further enhance antitumor immune responses in addition to direct cytotoxic effects. We therefore investigated whether MIV-818 induces similar immunomodulating effects as single agent or in combination with pembrolizumab. Methods: Immune-mediated tumor cell killing was determined by co-culturing labelled SKOV3 cancer cells with peripheral blood mononuclear cells (PBMC) and quantified over time using the IncuCyte ZOOM system. Effects on inflamed host tumor microenvironments (TMEs) were investigated by cytokine profiling co-cultures of activated PBMCs, primary fibroblasts or endothelial cells and HT29 colon adenocarcinoma cells. Effects on Staphylococcal enterotoxin B (SEB)-stimulated PBMC were determined by quantifying thymidine incorporation, and cytokine expression in supernatants. Results: Co-incubation of SKOV3 and PBM cells with MIV-818 induced a substantially higher level of apoptosis in the cancer cells (74±25%) compared to co-incubation in the absence of MIV-818 (37±23%). MIV-818 inhibited the proliferation of SKOV-3 cells in the absence PBMCs, but with no significant induction of apoptosis (IC50 7.5nM; ≤5% apoptosis). In a more complex model of the immune TME, MIV-818 treatment impacted inflammatory and angiogenesis-related responses in both the stromal and vascular co-culture systems, increasing IFNγ, IL-10, IL-17A, and TNFα in a manner comparable to that of pembrolizumab alone while also inhibiting IL-6. Combining MIV-818 with pembrolizumab further enhanced an immune-stimulatory effect by increasing IL-10, IL-17A and IFNy, while also modulating immune-related (GranB, IL-2, IL-6), and inflammation-related cytokines (VCAM-1, IP-10, TNFα). MIV-818 demonstrated dose-dependent inhibitory effect on proliferation of SEB-stimulated PBMCs. Despite this growth inhibitory effect, it led to a strong, dose-dependent, increase in IL-2 levels as a single agent which was further enhanced in combination with pembrolizumab. Conclusions: MIV-818 exerts favorable immunomodulating effects in vitro by increasing immune cytokine levels and the PBMC-mediated killing of cancer cells. These effects are further enhanced in combination with pembrolizumab and support future combination of MIV-818 with PD-(L)1 inhibitors in patients with HCC and other liver cancers. The effects of MIV-818 on tumor immune activation markers are being assessed the ongoing phase 1/2 clinical trial. Citation Format: Fredrik G Oberg, Karin Tunblad, Ana Slipicevic, Richard Bethell, Mark R Albertella. MIV-818 stimulates an antitumor immune response in vitro and enhances the effects of pembrolizumab [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C056. doi:10.1158/1535-7163.TARG-19-C056

CC-92480 Is a Novel Cereblon E3 Ligase Modulator with Enhanced Tumoricidal and Immunomodulatory Activity Against Sensitive and Resistant Multiple Myeloma Cells

Lenalidomide- and pomalidomide-based therapies are effective drugs in the treatment of patients with multiple myeloma (MM), however most patients with MM eventually relapse or become resistant. CC-92480, a novel cereblon (CRBN) E3 ligase modulator (CELMoD) with multiple activities including potent immunomodulation and single-agent antiproliferative effects, is being investigated in a phase 1 clinical trial (CC-92480-MM-001; NCT03374085) for patients with relapsed/refractory MM (RRMM). The present study investigates the preclinical data and mechanism of action of CC-92480 in MM models. CELMoD agents bound to CRBN confer differentiated substrate-degradation specificity on the CRL4CRBN E3 ubiquitin ligase. CRBN-modulator agents mediate destruction of Ikaros and Aiolos, transcription factors that contribute to myeloma cell survival. CC-92480 was found to produce rapid, deep, and sustained degradation of Ikaros and Aiolos, with superior antimyeloma activity. Accordingly, in a CRBN protein competitive binding assay, CC-92480 displaced a Cy-5-labeled CELMoD analog from CRBN with a 50% inhibitory concentration (IC50) value of 0.03 μM, whereas lenalidomide competed with an IC50 value of 1.27 μM in the same assay, demonstrating a higher binding affinity of CC-92480 for CRBN. Additionally, CC-92480 promoted the recruitment of Ikaros to the CRBN E3 ligase complex more effectively than pomalidomide in 2 orthogonal CRBN/Ikaros binding assays; it also triggered a more extensive cellular ubiquitination of Ikaros, and a faster, more efficient depletion of cellular Ikaros and Aiolos than pomalidomide. In various MM cell lines, including those with acquired resistance to lenalidomide or pomalidomide and low levels of CRBN, CC-92480 produced robust degradation of Ikaros and Aiolos followed by strong reduction of 2 additional and highly critical transcription factors, c-Myc and interferon regulatory factor 4, which are linked to the induction of apoptosis as measured by cleaved caspase-3. The tumoricidal activity of CC-92480 was shown to be CRBN dependent, since the effect was prevented by complete loss of CRBN or by the stabilization of Ikaros and Aiolos. CC-92480 displayed broad and potent antiproliferative activity across a panel of 20 MM cell lines that are either sensitive, have acquired resistance, or are refractory to lenalidomide or pomalidomide; the cell lines also contained diverse chromosomal translocations and oncogenic drivers typically found in MM patients. Approximately half of the MM cell lines evaluated were highly sensitive to CC-92480, with IC50 values for antiproliferative activity ranging from 0.04 to 5 nM; only 2 cell lines had IC50 values &gt; 100 nM. CC-92480 inhibits cell proliferation and induces apoptosis in MM cell lines that are not sensitive to lenalidomide or pomalidomide. This panel of cell lines includes both refractory cell lines and resistant cell lines generated through continuous exposure to lenalidomide and pomalidomide that acquired low levels of CRBN protein or mutations in the CRBN gene. CC-92480 also induced deep destruction of Ikaros and Aiolos in cultures of peripheral blood mononuclear cells (PBMCs), which led to the activation of T cells and increased production of the cytokines interleukin-2 and interferon gamma. These responses occurred at the range of CC-92480 concentrations that show potent tumoricidal effect against MM cells. The T cell activation and enhanced cytokine production by CC-92480 led to the potent and effective immune-mediated killing of MM cells in co-cultures with PBMCs. CC-92480 is a potent antiproliferative and proapoptotic novel CELMoD with enhanced autonomous cell-killing activity in MM cells that are either sensitive, resistant, or have acquired resistance to lenalidomide and pomalidomide. CC-92480 has a unique and rapid degradation profile stemming from the enhanced efficiency to drive the formation of a protein-protein interaction between Ikaros and Aiolos and CRBN, inducing cytotoxic effects in a CRL4CRBN-dependent fashion that leads ultimately to the induction of apoptosis, even in the context of low or mutated CRBN protein. Additionally, similar to lenalidomide, CC-92480 conserves immunomodulatory activity against MM cells. These data support the clinical investigation of CC-92480 in patients with RRMM. Disclosures Lopez-Girona: Celgene Corporation: Employment. Havens:Pfizer: Employment, Equity Ownership; Celgene: Equity Ownership. Lu:Celgene Corporation: Employment, Equity Ownership. Rychak:Celgene Corporation: Employment, Equity Ownership. Mendy:Celgene Corporation: Employment. Gaffney:Celgene: Employment. Surka:Celgene: Employment, Equity Ownership. Lu:Celgene Corporation: Employment, Equity Ownership. Matyskiela:Celgene corporation: Employment. Khambatta:Celgene: Employment. Wong:Celgene Corporation: Employment, Equity Ownership. Hansen:Celgene Corporation: Employment. Pierce:Celgene Corporation: Employment, Equity Ownership. Cathers:Global Blood Therapeutics (GBT): Employment; Celgene Corporation: Equity Ownership. Carmichael:Celgene plc: Employment, Equity Ownership.