Increased Antitumor Activity Research Articles

Phase I study of AMG 757, a half-life extended bispecific T-cell engager (HLE BiTE immune therapy) targeting DLL3, in patients with small cell lung cancer (SCLC).

TPS9080 Background: SCLC is an aggressive neuroendocrine tumor with poor prognosis and few treatment options. Delta-like ligand 3 (DLL3) is an inhibitory Notch ligand that is highly expressed on the surface of most SCLC tumors but minimally expressed in normal tissues. As such, DLL3 may be a promising therapeutic target. AMG 757 is an HLE BiTE immune therapy designed to redirect cytotoxic T cells to cancer cells by binding to DLL3 on cancer cells and CD3 on T cells, resulting in T cell activation and expansion and T cell-dependent killing of tumor cells. In addition to its direct antitumor effect, BiTE immune therapy can inflame the tumor microenvironment. Combining AMG 757 with a PD-1 pathway inhibitor may lead to increased antitumor activity by enabling sustained T cell-dependent killing of tumor cells. Methods: NCT03319940 is an open-label, ascending, multiple-dose, phase 1 study evaluating AMG 757 as monotherapy; the protocol was recently amended to also evaluate AMG 757 in combination with pembrolizumab. The study will include a dose exploration (monotherapy and combination) followed by a dose expansion (monotherapy). Key eligibility criteria: adult patients with relapsed/refractory SCLC whose disease progressed or recurred after at least 1 platinum-based chemotherapy regimen, ECOG performance status 0–2, at least 2 measurable lesions per modified RECIST 1.1, no untreated or symptomatic brain metastases, and adequate organ function. Primary objectives are to evaluate safety/tolerability and determine the maximum tolerated dose or recommended phase 2 dose of AMG 757 as monotherapy and in combination with pembrolizumab. Secondary objectives are to characterize pharmacokinetics and evaluate preliminary antitumor activity; exploratory objectives are to assess immunogenicity and changes in biomarkers in blood and tumor tissue. In the dose exploration phase, dose escalation/de-escalation decisions will be guided by a Bayesian logistic regression model; backfill enrollment at dose levels deemed safe and tolerable will be allowed. The study is open and recruiting patients. Clinical trial information: NCT03319940.

Interim subgroup analysis for response by PD-L1 status of CLASSICAL-Lung, a phase Ib/II study of pepinemab (VX15/2503) in combination with avelumab in advanced NSCLC.

3011 Background: Antibody blockade of semaphorin 4D (SEMA4D, CD100) promotes tumoral dendritic cell and CD8+ T cell infiltration and reduces function and recruitment of immunosuppressive myeloid cells. Importantly, these mechanisms to overcome immune exclusion and suppression have been shown to complement immune checkpoint therapies in preclinical models. Pepinemab is an IgG4 humanized monoclonal antibody targeting semaphorin 4D. The CLASSICAL-Lung clinical trial tests the combination of pepinemab with avelumab to couple T cell activation via checkpoint inhibition with beneficial modifications of the immune microenvironment via pepinemab. Methods: This phase 1b/2, single arm, first-in-human study is designed to evaluate the safety, tolerability and efficacy of pepinemab with avelumab in 62 patients (pts) with advanced (stage IIIB/IV) non-small cell lung cancer (NSCLC), including immunotherapy-naïve (ION) pts and pts whose tumors progressed following immunotherapy (IOF). Results: Among 21 evaluable ION pts, 5 experienced partial response (PR), 3 pts had clinical benefit ≥ 1 year, and the disease control rate (DCR) is 81%. Pts enrolled in this study were observed to have lower PD-L1 expression relative to prior single agent studies (likely due to approval of pembrolizumab for first line therapy). We, therefore, performed subgroup analysis for response by PD-L1 status. The objective tumor response (ORR) in the PD-L1 negative and low population ( < 80% TPS by Dako 73-10 assay) appears to be approximately 2-2.5 fold greater with combination therapy than with historical single agent immune checkpoint controls. Notably, 97% of pts who experienced PR or SD were reported to have tumors with negative or low PD-L1 expression. Among 29 evaluable IOF pts, the combination resulted in 59% DCR, including 2 PR and 7 patients with durable clinical benefit of ≥ 23 weeks. Biomarker analysis of pre- and on-treatment biopsies confirmed increased CD8+ T cell density correlating with response. Surprisingly, analysis of myeloid-derived suppressor cells (MDSCs) revealed a relative paucity of these cells in pretreatment NSCLC biopsies as compared to other cancer indications such as HNSCC. Conclusions: This trial is nearing completion with only 5 of 62 subjects remaining on study. Preliminary data suggest the combination is well tolerated and shows signs of increased antitumor activity, particularly in PD-L1 negative or low tumors. Updated clinical response data and immunophenotypic analyses will be presented. Clinical trial information: NCT03268057 .

Abstract B23: Insight towards therapeutic susceptibility of KRAS mutant cancers from MRTX1257: A prototype selective inhibitor of KRAS G12C

Abstract KRAS G12C is a driver mutation and the most frequent KRAS mutation in lung cancer. The ability to effectively target mutated KRAS has remained elusive despite decades of research. A structure-based drug design discovery program identified mutant-selective, covalent inhibitors of KRAS G12C with low nanomolar cell potency and favorable oral drug properties. MRTX1257 is a research tool compound that demonstrates selective irreversible modification of KRAS G12C and inhibits ERK1/2 phosphorylation with an IC50 value of 1 nM in a H358 cell-based assay. MRTX1257 was evaluated in 3D ultra-low adherent (ULA) viability assays across a panel of KRAS G12C mutated cancer cell lines in vitro and inhibited the growth of 16 out of 17 KRAS G12C-mutant cell lines tested with IC50 values ranging from 0.3 to 62 nM. MRTX1257 was inactive in non-KRAS G12C-mutant cell lines. Oral administration of MRTX1257 to mice bearing H358 xenograft tumors demonstrated a dose-dependent modification of KRAS G12C-mutant protein and inhibition of ERK1/2 and S6 phosphorylation. MRTX1257 also demonstrated dose-dependent KRAS target modification and cytoreductive antitumor efficacy in the KRAS G12C-mutant MIA PaCa-2 xenograft model with durable complete regressions. MRTX1257 was evaluated across an additional large panel of KRAS G12C-mutant subcutaneous cell-derived and patient-derived xenografts and demonstrated broad-spectrum antitumor activity including regressions greater than 30% in 18 out of 23 models tested. A small subset of models, exemplified by the H2122 model, demonstrated rapid initial tumor regression, followed by tumor stasis, suggesting that there may be mechanisms of adaptive tolerance to MRTX1257 treatment. Consistent with drug tolerance kinetics, MAP kinase pathway reactivation was observed as evidenced by downregulation of dual specificity phosphatases (DUSPs) and rebound of pERK and pS6 signaling during continuous treatment. To further elucidate mechanisms of drug tolerance and to identify combination strategies that could address feedback signaling, we tested MRTX1257 together with ~70 rationally selected compounds across a panel of genetically characterized cell lines. Combination treatment with a pan-EGFR family inhibitor more fully inhibited downstream signaling in vitro and led to increased antitumor activity in some models in vivo. A subset of KRAS G12C mutation-positive tumors may exhibit heterogenous molecular characteristics and mechanisms of reactivating KRAS dependent signaling, which is susceptible to combinatorial treatment strategies. Together, these data indicate the therapeutic susceptibility and broad dependence of KRAS G12C mutation-positive tumors on KRAS for tumor cell growth and survival and support the feasibility of developing mutant-selective small molecules. Citation Format: Jill Hallin, Ruth Aranda, Brian R. Baer, David M. Briere, Michael R. Burkhard, Andrew Calinisan, Harrah Chiang, Lars D. Engstrom, Jay B. Fell, John P. Fischer, Lauren Hargis, Matthew A. Marx, Pete Olson, Niranjan Sudhakar, James G. Christensen. Insight towards therapeutic susceptibility of KRAS mutant cancers from MRTX1257: A prototype selective inhibitor of KRAS G12C [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B23.

A case of multi-agent drug resistant choriocarcinoma treated with Pembrolizumab

•High-risk multi-agent drug resistant GTN is a life threatening disease.•Majority of choriocarcinomas show intense PD-L1 immunoreactivity.•Pembrolizumab increases antitumor activity.•Effectiveness of Pembrolizumab in treating patients with high-risk multi-agent drug resistant GTN.

Abstract A30: Combined IDO inhibitor and OX40L-armed-oncolytic-adenovirus therapy improves survival in murine glioblastoma

Abstract Glioblastoma diagnoses associate with upregulation of the immunosuppressive indoleamine-2,3-dioxygenase (IDO) enzyme, which correlates with poor prognoses. IDO inhibitors are immunometabolic adjuvants that can reverse protumor immunosuppression through increasing effector T-cell metabolism. To further stimulate antitumor T-cell activation, the OX40L-armed-oncolytic-adenovirus (Delta-24-RGDOX) can be used, which has shown to induce immune mechanisms that activate antitumor cytotoxic properties of T cells. Thus, we hypothesized that combining IDO inhibitors and Delta-24-RGDOX synergizes to produce a therapeutic effect in glioblastoma. In this study, we used a syngeneic glioblastoma orthotopic immunocompetent model to test the efficacy of combining Delta-24-RGDOX and an IDO inhibitor (1MT or indoximod). Delta-24-RGDOX treatment resulted in a significant survival benefit compared to the control groups (mean survival, 46.5 vs. 38.5 days, P=0.02). Interestingly, the combination of Delta-24-RGDOX and 1MT produced a higher percentage of long-term survivors compared to single therapy with Delta-24-RGDOX (50% vs. 20%, P=0.03). Moreover, when these long-term glioblastoma survivors were rechallenged with the same glioblastoma cells, we observed 100% survival, indicating the establishment of immune memory by the combination therapy. Furthermore, functional studies showed a significant increase in antitumor activity of splenocytes from glioblastoma-bearing combination-treated mice compared to Delta-24-RGDOX-treated mice (P=0.009). Additionally, immunophenotyping by flow cytometry revealed that glioblastoma-bearing combination-treated mice yielded the highest levels of PD-1/TIM-3/CD8+ T cells compared to all other treatment groups and decreased levels of immune-tolerant populations, including regulatory T cells and myeloid-derived suppressor cells, compared to Delta-24-RGDOX-only treated mice, and correlated with complete tumor elimination. Most importantly, these data support the use of immunometabolic adjuvants combined with virotherapy as a treatment for glioblastoma to improve its dismal outcomes. Citation Format: Teresa T. Nguyen, Dong Ho Shin, Hong Jiang, Frederick Lang, Candelaria Gomez-Manzano, Juan Fueyo. Combined IDO inhibitor and OX40L-armed-oncolytic-adenovirus therapy improves survival in murine glioblastoma [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 A30.

Blockers of Nicotinic Acetylcholine Receptors Delay Tumor Growth and Increase Antitumor Activity of Mouse Splenocytes.

Blockade of α6, α3β2, α9α10, and α7 subtypes of nicotinic acetylcholine receptors slows tumor growth in vivo, increases cytotoxic activity of splenocytes from tumor-bearing mice, and, to some extent, reduces the viability of Ehrlich carcinoma cells in vitro. These data indicate that nicotinic acetylcholine receptors are involved in oncogenesis, affecting the survival of tumor cells, inter alia, via modulation of the antitumor immunity.

Delivery of Temozolomide and N3-Propargyl Analog to Brain Tumors Using an Apoferritin Nanocage.

Glioblastoma multiforme (GBM) is a grade IV astrocytoma, which is the most aggressive form of brain tumor. The standard of care for this disease includes surgery, radiotherapy and temozolomide (TMZ) chemotherapy. Poor accumulation of TMZ at the tumor site, tumor resistance to drug, and dose-limiting bone marrow toxicity eventually reduce the success of this treatment. Herein, we have encapsulated >500 drug molecules of TMZ into the biocompatible protein nanocage, apoferritin (AFt), using a "nanoreactor" method (AFt-TMZ). AFt is internalized by transferrin receptor 1-mediated endocytosis and is therefore able to facilitate cancer cell uptake and enhance drug efficacy. Following encapsulation, the protein cage retained its morphological integrity and surface charge; hence, its cellular recognition and uptake are not affected by the presence of this cargo. Additional benefits of AFt include maintenance of TMZ stability at pH 5.5 and drug release under acidic pH conditions, encountered in lysosomal compartments. MTT assays revealed that the encapsulated agents displayed significantly increased antitumor activity in U373V (vector control) and, remarkably, the isogenic U373M (MGMT expressing TMZ-resistant) GBM cell lines, with GI50 values <1.5 μM for AFt-TMZ, compared to 35 and 376 μM for unencapsulated TMZ against U373V and U373M, respectively. The enhanced potency of AFt-TMZ was further substantiated by clonogenic assays. Potentiated G2/M cell cycle arrest following exposure of cells to AFt-TMZ indicated an enhanced DNA damage burden. Indeed, increased O6-methylguanine (O6-MeG) adducts in cells exposed to AFt-TMZ and subsequent generation of γH2AX foci support the hypothesis that AFt significantly enhances the delivery of TMZ to cancer cells in vitro, overwhelming the direct O6-MeG repair conferred by MGMT. We have additionally encapsulated >500 molecules of the N3-propargyl imidazotetrazine analog (N3P), developed to combat TMZ resistance, and demonstrated significantly enhanced activity of AFt-N3P against GBM and colorectal carcinoma cell lines. These studies support the use of AFt as a promising nanodelivery system for targeted delivery, lysosomal drug release, and enhanced imidazotetrazine potency for treatment of GBM and wider-spectrum malignancies.

Trifluridine/tipiracil in combination with oxaliplatin and either bevacizumab or nivolumab: Results of the expansion part of a phase I study in patients with metastatic colorectal cancer.

140 Background: Trifluridine/tipiracil (FTD/TPI) is approved for use in patients (pts) with pretreated mCRC. In vivo studies have shown an increase in anti-tumor activity when combining FTD/TPI, oxaliplatin or bevacizumab, and an increase in tumor immunogenicity after treatment with FTD/TPI and oxaliplatin (Ghiringhelli, 2018). The recommended dose for expansion had been defined as FTD/TPI 35 mg/m² bid, days 1–5 q14, together with oxaliplatin 85 mg/m² (day 1). Methods: In addition to FTD/TPI and oxaliplatin, eligible pts received bevacizumab 5 mg/kg (Cohort A) or nivolumab 3 mg/kg (Cohort B) on day 1. Eligibility criteria included measurable disease, performance status (PS) 0-1, normal organ function, progression after &gt; 1 prior anti-tumor therapy (excluding oxaliplatin), and confirmed MSS status (Cohort B). A Bayesian design was used for futility and efficacy assessments. Efficacy endpoints of objective response rate (ORR), disease control rate (DCR), progression free survival (PFS) and adverse events (AEs) were assessed for each cohort; biomarkers of immune function including PD-L1 expression for Cohort B. Results: A total of 37 and 17 pts were enrolled in Cohorts A and B respectively; with a median age of 64 years (range 33 to 83 years), 61% and 39% had an PS of 0 and 1. At baseline, no pt showed PD-L1 expression on tumor cells, and only 1 pt on immune cells (5% threshold). At data cutoff, 32 and 12 pts were evaluable for response in Cohorts A and B. In Cohort A, ORR was 13% (1 CR; 3 PR), and DCR was 91%. The mPFS was 6.9 months (95% IC, 4.3-9.3). In Cohort B, ORR was 8% (1 PR), DCR was 67%. The mPFS was 6.5 months (95% IC, 1.8-8.6). Overall, the most common treatment-related AEs (≥20% of pts) included neutropenia, nausea, diarrhoea, and fatigue; only 1 pt reported grade 3 febrile neutropenia; 5 pts discontinued due to AEs and no treatment-related death were reported. Conclusions: In this study, bevacizumab in addition to FTD/TPI and oxaliplatin showed antitumor activity. The cohort of nivolumab was prematurely discontinued due to lack of efficacy. Both cohorts showed an acceptable safety profile in pretreated mCRC pts. Clinical trial information: NCT02848443.

Objective Response Rate Among Patients With Locally Advanced or Metastatic Sarcoma Treated With Talimogene Laherparepvec in Combination With Pembrolizumab

Patients with advanced sarcoma have limited treatment options. Talimogene laherparepvec (T-VEC) has been shown to increase tumor-specific immune activation via augmenting antigen presentation and T-cell priming. To examine whether T-VEC in combination with pembrolizumab is associated with increased tumor-infiltrating lymphocyte infiltration and programmed death-ligand 1 expression and thus with increased antitumor activity in patients with locally advanced or metastatic sarcoma. This open-label, single-institution phase 2 interventional trial of T-VEC plus pembrolizumab enrolled 20 patients with locally advanced or metastatic sarcoma between March 16 and December 4, 2017, for whom at least 1 standard systemic therapy had failed. The median duration of therapy was 16 weeks (range, 7-67 weeks). Reported analyses include data through December 14, 2018. Patients received pembrolizumab (200-mg flat dose) intravenously and T-VEC (first dose, ≤4 mL × 106 plaque-forming units [PFU]/mL; second and subsequent doses, ≤4 mL × 108 PFU/mL) injected into palpable tumor site(s) on day 1 of each 21-day cycle. The primary end point was objective response rate (ORR; complete response and partial response) at 24 weeks determined by Response Evaluation Criteria In Solid Tumors (RECIST), version 1.1, criteria. Secondary end points included best ORR by immune-related RECIST criteria, progression-free survival rate at 24 weeks, overall survival, and safety. All 20 patients (12 women [60%]; median age, 63.5 years [range, 24-90 years]) were evaluable for response. The study met its primary end point of evaluating the best ORR at 24 weeks determined by RECIST, version 1.1, criteria; the best ORR was 30% (95% CI, 12%-54%; n = 6). The ORR overall was 35% (95% CI, 15%-59%; n = 7). The incidence of grade 3 treatment-related adverse events was low (4 patients [20%]). There were no grade 4 treatment-related adverse events or treatment-related deaths. In this phase 2 clinical trial, treatment with T-VEC plus pembrolizumab was associated with antitumor activity in advanced sarcoma across a range of sarcoma histologic subtypes, with a manageable safety profile. This combination therapy met its predefined primary study end point; further evaluation of T-VEC in combination with pembrolizumab for patients with select sarcoma subtypes is planned. ClinicalTrials.gov identifier: NCT03069378.

Enterotoxins can support CAR T cells against solid tumors

Responses of solid tumors to chimeric antigen receptor (CAR) T cell therapy are often minimal. This is potentially due to a lack of sustained activation and proliferation of CAR T cells when encountering antigen in a profoundly immunosuppressive tumor microenvironment. In this study, we investigate if inducing an interaction between CAR T cells and antigen-presenting cells (APCs) in lymphoid tissue, away from an immunosuppressive microenvironment, could enhance solid-tumor responses. We combined CAR T cell transfer with the bacterial enterotoxin staphylococcal enterotoxin-B (SEB), which naturally links a proportion of T cell receptor (TCR) Vβ subtypes to MHC-II, present on APCs. CAR T cell proliferation and function was significantly enhanced by SEB. Solid tumor-growth inhibition in mice was increased when CAR T cells were administered in combination with SEB. CAR T cell expansion in lymphoid tissue was demonstrated, and inhibition of lymphocyte egress from lymph nodes using FTY720 abrogated the benefit of SEB. We also demonstrate that a bispecific antibody, targeting a c-Myc tag on CAR T cells and cluster of differentiation 40 (CD40), could also enhance CAR T cell activity and mediate increased antitumor activity of CAR T cells. These model systems serve as proof-of-principle that facilitating the interaction of CAR T cells with APCs can enhance their ability to mediate antitumor activity.

Extremely Effective Chemoradiotherapy by Inducing Immunogenic Cell Death and Radio-Triggered Drug Release under Hypoxia Alleviation.

Local hypoxia in solid malignancies often results in resistance to radiotherapy (RT) and chemotherapy (CT), which may be one of the main reasons for their failure in clinical application. Especially, oxygen is an essential element for enhancing DNA damage caused by ionizing radiation in radiotherapy. Here, two biomimetic oxygen delivery systems were designed by encapsulating hemoglobin (Hb) alone into a liposome (Hb-Lipo) or co-encapsulating Hb and doxorubicin (DOX) into a liposome (DOX-Hb-Lipo). Our data indicated that both Hb-Lipo and DOX-Hb-Lipo could effectively alleviate hypoxia in tumors. We demonstrated that RT plus tumor-targeting delivery of oxygen mediated by Hb-Lipo could significantly overcome the tolerance of hypoxic cancer cells to RT, showing significantly enhanced cancer-cell killing and tumor growth inhibition ability, mainly attributing to hypoxia alleviation and increased reactive oxygen species production under RT in cancer cells. Furthermore, a melanoma model that was quite insensitive to both RT and CT was used to test the efficacy of chemoradiotherapy combined with hypoxia alleviation. RT plus Hb-Lipo only caused a limited increase in antitumor activity. However, extremely strong tumor inhibition could be obtained by RT combined with DOX-Hb-Lipo-mediated CT, attributed to radio-triggered DOX release and enhanced immunogenic cell death induced by RT under an oxygen supplement. Our study provided a valuable reference for overcoming hypoxia-induced radioresistance and a useful therapeutic strategy for cancers that are extremely insensitive to chemo- or radiotherapy.

Preparation of Quercetin Loaded Microparticles and their Antitumor Activity against Human Lung Cancer Cells (A549) in vitro

Novel quercetin-loaded microparticles (QM) were fabricated using coaxial electrospraying, characterized for surface morphology and release profile, and evaluated for antitumor activity in vitro. QM exhibited an average diameter of 1.69 ±1.13 mm, which was an appropriate size suitable for respiratory delivery. X-ray diffraction patterns showed that the components in QM existed in an amorphous physical form, leading to favorable interactions between the drug (quercetin), the polymer matrix (polyvinylpyrrolidone, PVP) and other excipients (sodium dodecyl sulfate and sucralose). QM performed much faster release rate compared with free quercetin powder (Q) in vitro. Furthermore, QM also showed more potent inhibitory effects on A549 cell growth with reduced cell viability, decreased cell migration and induced more G0/G1 phase cell cycle arrest than Q. Thus, the quercetin loaded microparticles exhibited more potent inhibitory effects than free quercetin on A549 cell. The increased antitumor activity could be attributed to the enhanced accumulation of quercetin in the A549 cells with the QM. However, further studies are necessary to elucidate the exact mechanisms.

Biodegradable pH-sensitive prospidine-loaded dextran phosphate based hydrogels for local tumor therapy

The paper focuses on the development of drug delivery systems based on hydrogels of dextran phosphate (DP) for local cancer therapy. The hydrogels were characterized by physicochemical properties including functional group content, morphology, gel fraction, pH-responsive swelling. The desirable pH-sensitive drug release behavior of these hydrogels was demonstrated by a drug release test with Prospidine-loaded hydrogels (DP-Pr hydrogels) at different pH values. In vitro degradation of the DP-Pr hydrogels was determined under simulated physiological conditions. The cytotoxicity of the blank DP hydrogels and DP-Pr hydrogels with different Pr concentrations was evaluated with HeLa and HЕр-2 cells. Investigations of antitumor efficiency in vivo showed that administration of DP-Pr hydrogels in comparison with an aqueous solution of Pr results in the increase of antitumor activity, prolongation of therapeutic action and growth of a number of animals cured. Therefore, such pH-responsive DP hydrogels could be promising candidates as drug delivery carriers.

Reactive Oxygen Species in the Tumor Microenvironment: An Overview.

Reactive oxygen species (ROS) are important signaling molecules in cancer. The level of ROS will determine physiological effects. While high levels of ROS can cause damage to tissues and cell death, low levels of ROS can have a proliferative effect. ROS are produced by tumor cells but also cellular components that make up the tumor microenvironment (TME). In this review, we discuss the mechanisms by which ROS can affect the TME with particular emphasis on tumor-infiltrating leukocytes. Greater insight into ROS biology in this setting may allow for therapeutic manipulation of ROS levels in order to remodel the tumor microenvironment and increase anti-tumor activity.

Recombinant Adenovirus Expressing a Soluble Fusion Protein PD-1/CD137L Subverts the Suppression of CD8+ T Cells in HCC.

Oncolytic viruses are an excellent platform for developing effective strategies in cancer immunotherapy. Several challenges remain in the use of viro-immunotherapy for cancer, such as the lack of costimulatory signals and negative regulation of immune checkpoints. In this study, we designed a novel adenovirus expressing a soluble fusion protein, programmed cell death protein 1 (PD-1)/CD137L, which contains the extracellular domains of PD-1 and CD137L at each terminus (Ad5-PC). Ad5-PC preserved the costimulatory activity of CD137L and facilitated the persistence of activated CD8+ Tcells. Ad5-PC induced strikingly increased antitumor activity in both ascitic and subcutaneous hepatocellular carcinoma (HCC) tumor models, with 70% and 60% long-term cure rates, respectively. The improved antitumor effect of Ad5-PC was attributed to the sustained high-level lymphocyte activation and interferon (IFN)-γ production in the tumor microenvironment, and was essentially dependent on CD8+ Tcells rather than natural killer (NK) cells. Moreover, Ad5-huPC-expressing human soluble PD-1/CD137L fusion protein was effective in suppressing tumor growth and improving survival in a humanized mouse model. We confirmed that Ad5-PC induced tumor-specific and systematic protection against tumor rechallenges at both in situ and distant sites. Thus, Ad5-PC harnesses several distinct functions to efficiently overcome several major hurdles of viro-immunotherapy.

Ex vivo expansion of autologous, donor-derived NK-, γδT-, and cytokine induced killer (CIK) cells post haploidentical hematopoietic stem cell transplantation results in increased antitumor activity.

Posttransplant treatment strategies are narrowed by the vulnerability of bone marrow. Building on immune cells with antitumor activity is a growing field in cancer therapy. Thus, transfer of expanded and preactivated immune cells is a promising intensification of treatment in high-risk tumor patients. We tested ex vivo expanded NK-, γδT-, and CIK cells that were generated by coincubation with irradiated K562-mb15-41BBL and Il2 and compared the expansion conditions of PBMCs versus CD3-depleted PBMCs as well as static versus semi-automated expansion. The median fold expansion was significantly higher using PBMCs and static expansion conditions. Expanded cells were preactivated with a CD56brightCD69high immunophenotype exerting excellent direct cellular cytotoxicity as well as ADCC in various tumor entities. We established a large-scale clinical-grade ex vivo expansion and activation protocol of NK-, γδT-, and CIK cells from donor-derived PBMCs of patients after haploidentical HSCT. In a patient with AML, NK/γδT/CIK cell transfer was associated with MRD response. A significant increase of direct antitumor activity and ADCC post cell transfer was documented. The results that we report here provide the rationale for clinical testing of expanded, preactivated NK/γδT/CIK cells for cancer therapy.

Abstract 1963: Feasibility and efficacy of using self-delivering RNAi against TGFB1 to reduce TME immunosuppression

Abstract One of the leading causes of non-responding tumors with existing immunotherapies is an immunosuppressive tumor micro-environment (TME). The suppressive nature of the TME is, in part, mediated by immunoregulatory cells (e.g. MDSC cells) and cytokines/chemokines. TGFB1 has recently been shown to be a key suppressive factor responsible for facilitating “cold” or immune-excluded tumors. In these tumors, T cell activity is restricted and by reducing TGFB1 levels we aim to support anti-tumor T cell activity. We evaluated if downregulation of TGFB1 with a self-delivering RNAi (sd-rxRNA) in MDSCs (in vitro) or TME (in vivo) promotes T cell activity and increase anti-tumor activity. sd-rxRNA is a chemically modified RNAi which incorporates drug like properties allowing for self-delivery of RNAi compounds to immune cells (including MDSC, DC, T and NK cells) in the absence of electroporation or formulation in a delivery vehicle. Tumor induced MDSCs were incubated with sd-rxRNA targeting TGFB1, its downstream target COX2 or in combination for 2 days. The cells were washed and added to a co-culture with OT-1 T cells and OVA expressing B16 melanoma. The anti-tumor activity of T-cells was monitored using a Real-Time Cell Activity assay (RTCA). TGFB1 and COX2 downregulated MDSCs had reduced immune-suppressive activity as shown by the enhanced B16-OVA tumor cell killing by CD8 OT-1 T cells in the RTCA assay while the combination of TGFB1 and COX2 MDSCs did not further improve the anti-tumor activity. To evaluate the effect of anti-TGFB1 sd-rxRNA in vivo, we first studied the tumor distribution and TGFB1 downregulation by intratumoral injection of sd-rxRNA to immunocompetent Balbc mice bearing 4T1-RL-GFP breast cancer in a fat pad model. DY547 labeled sd-rxRNA targeting PPIB or unlabeled sd-rxRNA targeting TGFB1 was injected into tumors. Two days later, tumors were resected, and fluorescence signal was analyzed by microscopy and TGFB1 downregulation was evaluated by RT-PCR. Efficient delivery of the DY547 labeled sd-rxRNA was observed in the vast majority of cells as shown by fluorescence microscopy. In addition, fluorescence signal could be detected in tumors up to 28 days after intratumoral injection, suggesting that sd-rxRNA was stable in tumors. Downregulation of TGFB1 in resected tumors, and an increased anti-tumor activity, was seen after three injections of sd-rxRNA. A follow up study was initiated investigating the efficacy of TGFB1 sd-rxRNA in combination with local tumor radiation on anti-tumor and metastatic activities in a 4T1 orthotopic breast cancer model. Preliminary data shows promising results. Our results suggest that sd-rxRNA compounds targeting TGFB1 can enhance the anti-tumor activity of T cells. Local injection of sd-rxRNA into tumors can effectively target TGFB1 in the TME resulting in less immunosuppression and increased anti-tumoral activity. Citation Format: Winnie F. Tam, Dingxue Yan, Melissa Maxwell, James Cardia, Gerrit Dispersyn. Feasibility and efficacy of using self-delivering RNAi against TGFB1 to reduce TME immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1963.

A phase I, first-in-human, open label, dose-escalation and cohort expansion study of MGD019, a bispecific DART protein binding PD-1 and CTLA-4 in patients with unresectable or metastatic neoplasms.

TPS2661 Background: Immune checkpoint molecules, including CTLA-4 and PD-1, attenuate the duration and strength of adaptive immune responses to limit immune-mediated tissue damage. Tumors may inhibit cellular immune activation by expressing ligands that bind checkpoint molecules and inhibit T-cell function in the tumor microenvironment. Blockade of these inhibitory pathways is the primary mechanism of action of several novel cancer immunotherapy agents. Combined blockade of PD-1 and CTLA-4 with two checkpoint inhibitors, ipilimumab and nivolumab, increases antitumor activity beyond either single agent alone in patients with metastatic melanoma or other malignancies. MGD019, a novel bispecific molecule that co-engages and coordinately inhibits both PD-1 and CTLA-4 signaling, was developed to potentially improve antitumor activity and/or safety relative to the monoclonal antibody combination. MGD019 is an Fc-bearing tetravalent DART molecule (bivalent for each antigen) that can independently block either checkpoint molecule, with preferential co-blockade in cells co-expressing both molecules demonstrated in vitro. It is hypothesized that MGD019 might be clinically active in either checkpoint naïve or checkpoint experienced patients after prior PD-1/PD-L1 inhibitors. Methods: This Phase 1 study will characterize safety, dose limiting toxicities, and maximum tolerated dose (MTD)/maximum administered dose (MAD) of MGD019. Dose Escalation will enroll patients with advanced solid tumors of any histology in sequential escalating doses in cohorts of 3 to 9 patients in a 3+3+3 design. Once the MTD/MAD is reached, a Cohort Expansion phase will characterize safety and initial antitumor activity per RECIST v1.1 and irRECIST in patients with specific tumor types anticipated to be sensitive to dual checkpoint blockade. Additional endpoints include pharmacokinetics; immunogenicity; impact of MGD019 on various measures of immune-regulatory effects in peripheral blood and biopsy specimens; and relationship between antitumor activity and gene profiles, tumor mutational burden, and PD-1, PD-L1, and CTLA-4 expression on tumor cells and immune cell infiltrates within biopsy specimens. Patients will be followed for survival approximately every 3 months for 2 years. Clinical trial information: NCT03761017.

The multikinase inhibitor EC-70124 synergistically increased the antitumor activity of doxorubicin in sarcomas.

Cytotoxic drugs like doxorubicin remain as the most utilized agents in sarcoma treatment. However, advanced sarcomas are often resistant, thus stressing the need for new therapies aimed to overcome this resistance. Multikinase inhibitors provide an efficient way to target several pro-tumorigenic pathways using a single agent and may constitute a valuable strategy in the treatment of sarcomas, which frequently show an aberrant activation of pro-tumoral kinases. Therefore, we studied the antitumor activity of EC-70124, an indolocarbazole analog that have demonstrated a robust ability to inhibit a wide range of pro-survival kinases. Evaluation of the phospho-kinase profile in cell-of-origin sarcoma models and/or sarcoma primary cell lines evidenced that PI3K/AKT/mTOR, JAK/STAT or SRC were among the most highly activated pathways. In striking contrast with the structurally related drug midostaurin, EC-70124 efficiently prevented the phosphorylation of these targets and robustly inhibited proliferation through a mechanism associated to the induction of DNA damage, cell cycle arrest and apoptosis. In addition, EC-70124 was able to partially reduce tumor growth in vivo. Importantly, this compound inhibited the expression and activity of ABC efflux pumps involved in drug resistance. In line with this ability, we found that the combined treatment of EC-70124 with doxorubicin resulted in a synergistic cytotoxic effect in vitro and an increased antitumor activity of this cytotoxic drug in vivo. Altogether, these results uncover the capability of the novel multikinase inhibitor EC-70124 to counteract drug resistance in sarcoma and highlight its therapeutic potential when combined with current treatments.

Metformin increases antitumor activity of MEK inhibitor binimetinib in 2D and 3D models of human metastatic melanoma cells

Melanoma is one of the most aggressive and treatment-resistant tumors that responsible for majority of skin-cancer related deaths. Here we propose a combination of MEK inhibitor binimetinib with metformin as a promising therapy against human melanoma cells in vitro, including BRAF -mutated A375, Mel Z, and Mel IL cells, and NRAS-mutated Mel MTP and Mel Me cells. Additionally, we obtained two close to clinical practice models of melanoma progression. The first one was vemurafenib-resistant Mel IL/R melanoma cells with acquired resistance to BRAF inhibition-targeted therapy, and the second one was tumor spheroids, which are 3D in vitro model of small-size solid tumors in vivo. The cytotoxicity of binimetinib and metformin was synergistic in both 2D and 3D melanoma culture and mediated through apoptotic pathway. The combination reduced the number of melanoma-formed colonies, inhibited cell invasion and migration, and led to G0/G1 cell cycle arrest through cyclin D/CDK4/CDK6 pathway. The mechanism of metformin and binimetinib synergy in melanoma cells was associated with increased activation of p-AMPKα and decreased p-ERK, but not with alterations in p-mTOR. In summary, the combination of metformin and binimetinib resulted in stronger anti-proliferative effects on melanoma cells compared to binimetinib alone, and therefore could be promising for clinical applications.