Marked Tumor Research Articles

Feasibility of using a 99mTc-hydroxamamide complex containing an albumin binder moiety for in vivo albumin labeling-based tumor imaging

Human serum albumin (HSA), which is distributed throughout the blood, is used as a carrier for transporting drugs to tumors based on the enhanced permeability and retention (EPR) effect. To develop an agent for the in vivo radiolabeling of endogenous albumin, we designed and synthesized novel hydroxamamide (Ham)-based technetium-99m (99mTc) complexes, which contained a monovalent or bivalent 4-(4-iodophenyl)butyric acid (IA) derivative as an albumin binder (ALB) moiety ([99mTc]AB2 and [99mTc]ALB2, respectively), and evaluated their utility for in vivo tumor imaging. In an in vitro HSA-binding assay, [99mTc]AB2 and [99mTc]ALB2 showed greater binding to HSA than [99mTc]BHam, a 99mTc-Ham complex without an ALB moiety. In an in vivo biodistribution assay, [99mTc]ALB2 showed marked blood and tumor retention (25.13 and 4.61% injected dose (ID)/g, respectively, at 1 h postinjection), suggesting that the EPR effect had been induced. However, [99mTc]AB2 showed no marked blood or tumor retention (4.16 and 0.75% ID/g, respectively, at 1 h postinjection), probably because the affinity of the monovalent IA derivative for albumin was insufficient to induce the EPR effect. These findings indicated that the multivalent interactions of [99mTc]ALB2 had enhanced its affinity for albumin. 99mTc-complexes containing multivalent ALB moieties may be useful for tumor imaging.

Hepatic Cancer Stem Cells: Molecular Mechanisms, Therapeutic Implications, and Circulating Biomarkers.

Simple SummaryThe high heterogeneity of hepatocellular carcinoma (HCC), and the lack of druggable mutations, hamper the identification of unequivocal molecular classifiers and limit the discovery of selective therapeutic treatments. Moreover, the lack of circulating biomarkers guiding the choice of personalized treatments and identifying the occurrence of acquired resistance to treatments still represents an unmet clinical need in HCC. The cancer stem cell (CSC) compartment underlies tumor heterogeneity, disease recurrence, and drug resistance in all cancer types, including HCC. Knowledge of the molecular mechanisms supporting the maintenance and proliferation of CSCs may help to identify novel biomarkers and therapeutic targets to improve and refine HCC management.Hepatocellular carcinoma (HCC) is one of the deadliest cancers. HCC is associated with multiple risk factors and is characterized by a marked tumor heterogeneity that makes its molecular classification difficult to apply in the clinics. The lack of circulating biomarkers for the diagnosis, prognosis, and prediction of response to treatments further undermines the possibility of developing personalized therapies. Accumulating evidence affirms the involvement of cancer stem cells (CSCs) in tumor heterogeneity, recurrence, and drug resistance. Owing to the contribution of CSCs to treatment failure, there is an urgent need to develop novel therapeutic strategies targeting, not only the tumor bulk, but also the CSC subpopulation. Clarification of the molecular mechanisms influencing CSC properties, and the identification of their functional roles in tumor progression, may facilitate the discovery of novel CSC-based therapeutic targets to be used alone, or in combination with current anticancer agents, for the treatment of HCC. Here, we review the driving forces behind the regulation of liver CSCs and their therapeutic implications. Additionally, we provide data on their possible exploitation as prognostic and predictive biomarkers in patients with HCC.

A novel PLK1 inhibitor onvansertib effectively sensitizes MYC-driven medulloblastoma to radiotherapy.

Group 3 medulloblastoma (MB) is often accompanied by MYC amplification. PLK1 is an oncogenic kinase that controls cell cycle and proliferation and has been preclinically validated as a cancer therapeutic target. Onvansertib (PCM-075) is a novel, orally available PLK1 inhibitor, which shows tumor growth inhibition in various types of cancer. We aim to explore the effect of onvansertib on MYC-driven medulloblastoma as a monotherapy or in combination with radiation. Crisper-Cas9 screen was used to discover essential genes for MB tumor growth. Microarray and immunohistochemistry on pediatric patient samples were performed to examine the expression of PLK1. The effect of onvansertib in vitro was measure by cell viability, colony-forming assays, extreme limiting dilution assay, and RNA-Seq. ALDH activity, cell-cycle distribution, and apoptosis were analyzed by flow cytometry. DNA damage was assessed by immunofluorescence staining. Medulloblastoma xenografts were generated to explore the monotherapy or radio-sensitizing effect. PLK1 is overexpressed in Group 3 MB. The IC50 concentrations of onvansertib in Group 3 MB cell lines were in a low nanomolar range. Onvansertib reduced colony formation, cell proliferation, stem cell renewal and induced G2/M arrest in vitro. Moreover, onvansertib in combination with radiation increased DNA damage and apoptosis compared with radiation treatment alone. The combination radiotherapy resulted in marked tumor regression in xenografts. These findings demonstrate the efficacy of a novel PLK1 inhibitor onvansertib in vitro and in xenografts of Group 3 MB, which suggests onvansertib is an effective strategy as monotherapy or in combination with radiotherapy in MB.

Effect of Vitamin B2 and Vitamin E on Cancer-Related Sarcopenia in a Mouse Cachexia Model

Cancer-related sarcopenia is associated with impaired energy metabolism and increased oxidative stress production in skeletal muscles. With an aim to treat cancer-related sarcopenia using dietary intervention, we investigated the effects of vitamin B2 (VB2) and vitamin E (VE), which are recognized to have antioxidant effects, on CT26 mouse colon cancer cells and skeletal muscles in vitro and in vivo. VB2 suppressed tumor growth by suppressing cell proliferation and inducing more pronounced apoptosis by increasing the production of adenosine triphosphate (ATP) and reactive oxygen species (ROS). VE suppressed tumor growth by suppressing cell proliferation and increasing apoptosis by decreasing the production of ATP and ROS. In C2C12 mouse skeletal myoblast cells, VB2 treatment increased the production of ATP and ROS and VE treatment decreased the production of ATP and ROS; both treatments suppressed skeletal myoblast maturation. In the mouse model, intraperitoneal inoculation (peritoneal model) resulted in marked macrophage infiltration and elevated blood tumor necrosis factor-α and high-mobility group box-1 inflammatory cytokine levels, leading to cachexia. In contrast, subcutaneous inoculation (subcutaneous model) showed poor macrophage infiltration and low inflammatory cytokine levels, without cachexia. VB2 and VE activated macrophages and exacerbated cancer-related sarcopenia in the peritoneal model, whereas VB2 and VE treatment did not exhibit significant changes in sarcopenia in the subcutaneous model. In order to improve cancer-related sarcopenia by dietary intervention, it is important to consider the effect on inflammatory cytokines.

Targeted delivery of cytotoxic proteins to prostate cancer via conjugation to small molecule urea-based PSMA inhibitors

Prostate cancer cells are characterized by a remarkably low proliferative rate and the production of high levels of prostate-specific proteases. Protein-based toxins are attractive candidates for prostate cancer therapy because they kill cells via proliferation-independent mechanisms. However, the non-specific cytotoxicity of these potent cytotoxins must be redirected to avoid toxicity to normal tissues. Prostate-Specific Membrane Antigen (PSMA) is membrane-bound carboxypeptidase that is highly expressed by prostate cancer cells. Potent dipeptide PSMA inhibitors have been developed that can selectively deliver and concentrate imaging agents within prostate cancer cells based on continuous PSMA internalization and endosomal cycling. On this basis, we conjugated a PSMA inhibitor to the apoptosis-inducing human protease Granzyme B and the potent Pseudomonas exotoxin protein toxin fragment, PE35. We assessed selective PSMA binding and entrance into tumor cell to induce cell death. We demonstrated these agents selectively bound to PSMA and became internalized. PSMA-targeted PE35 toxin was selectively toxic to PSMA producing cells in vitro. Intratumoral and intravenous administration of this toxin produced marked tumor killing of PSMA-producing xenografts with minimal host toxicity. These studies demonstrate that urea-based PSMA inhibitors represent a simpler, less expensive alternative to antibodies as a means to deliver cytotoxic proteins to prostate cancer cells.

Abstract CT222: BrUOG360: A phase Ib/II study of copanlisib combined with rucaparib in patients with metastatic castration-resistant prostate cancer (mCRPC)

Abstract Background: Patients with metastatic prostate cancer invariably progress to a lethal state called metastatic castration-resistant prostate cancer (mCRPC). Approximately 23-28% of patients with mCRPC have molecular alterations in genes associated with homologous recombination (HR) DNA repair mechanisms (e.g., BRCA1/2). These alterations are associated with sensitivity to poly ADP-ribose polymerase inhibitors (PARPi). The responses to PARPi can be short-lived, and several mechanisms of resistance have been described. Preclinical studies demonstrated that PI3K suppression impairs HR and sensitizes cancer cells to PARPi even in the absence of mutations in DNA repair genes. The synergy between olaparib and PI3K inhibition led to marked tumor regression both in vitro and in vivo models of prostate cancer. These preclinical results, coupled with the importance of both the PI3K pathway and DNA damage repair mechanisms in mCRPC, support our hypothesis that dual inhibition of PI3K and PARP might improve clinical outcomes in mCRPC. We describe an ongoing phase Ib/II study aimed to evaluate the safety and determine the maximum tolerated dose (MTD) of the combination of copanlisib (pan-class I PI3K inhibitor) and rucaparib (a PARP-1, -2 and -3 inhibitor) followed by an expansion cohort exploring efficacy among patients with mCRPC. Methods: Phase I, dose-escalation part of the study, aims to establish the MTD and/or recommended phase II dose (RP2D) of copanlisib in combination with rucaparib. Dose escalation will follow a standard 3+3 escalation scheme. Evidence of HR-deficiency is not required to enroll in phase I, but is for phase II. All subjects will be treated with rucaparib (continuous oral administration twice daily) 400mg (dose level I), 500mg (II) or 600mg (III-IV) and intravenous copanlisib (D1, D8, D15; 28-day cycle; dose levels I-III: 45 mg; level IV: 60 mg). Once the RP2D of copanlisib and rucaparib in the phase I part of the study is determined, enrollment will commence into the phase II part of the trial. The primary objective of phase II is to estimate the preliminary efficacy of the combination of copanlisib and rucaparib among subjects with mCRPC carrying mutations in DNA repair genes, as measured by prostate-specific antigen (PSA) response. Up to 20 patients will enroll in the phase II using a Simon's two-stage design. In the first stage, 14 patients will be accrued. If there are 7 or fewer PSA responses in these 14 patients, the study will be stopped for futility. Otherwise, an additional 6 patients will be accrued. Patients with mCRPC need to have had disease progression during treatment with abiraterone, enzalutamide, and/or apalutamide. Prior therapy with taxanes is allowed. Translational objectives include the correlation of clinical outcomes with HR mutations and PI3K/mTOR pathway gene alterations in the tumor. Genomic analyses will also investigate biomarkers and mechanisms of action of the combination. The clinical trial is recruiting (NCT04253262). Citation Format: Andre De Souza, Rahul Aggarwal, Howard Safran, Dragan Golijanin, Roxanne Wood, Adam Olszewski, Wafik S. El-Deiry, Anthony Mega, Benedito A. Carneiro. BrUOG360: A phase Ib/II study of copanlisib combined with rucaparib in patients with metastatic castration-resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT222.

Multimodality Targeting of Dendritic Cell Vaccination Combined with Immunomodulatory Drug and Immune Checkpoint Blockade Has Synergistically Induced a Marked Tumor Regression By Modulating Tumor Microenvironment in a Murine Myeloma Model

There is emerging evidence that the maximal benefit of dendritic cell (DC)-based cancer immunotherapy may be achieved by combination with other therapies that act to immunomodulation and tumor microenvironment. In this study, we tried to obtain the best efficacy of immunotherapy using DC vaccination in combination with lenalidomide and PD-1 blockade in a murine myeloma model. After establishing myeloma-bearing mice, five treatment groups were designed to be a mimic protocol as like treatment in clinics as following: 1) PBS control, 2) DCs, 3) DCs + lenalidomide, 4) DCs + PD-1 blockade, and 5) DCs + lenalidomide + PD-1 blockade. After treatment, preclinical response and in vitro immunological responses were evaluated. DCs combined with lenalidomide and PD-1 blockade showed the best tumor regression among the study groups. These anti-tumor effects have meaningfully related to the decrease of immuno-regulatory populations, such as myeloid-derived suppressor cells (MDSCs), M2 macrophages, and regulatory T cells (Treg) and the increase of effector immune cell populations, including CD4+ and CD8+ T cells, natural killer (NK) cells, and M1 macrophages, accompanied with the activation of cytotoxic T lymphocytes (CTLs) and NK cells in the splenocytes from the treated mice. Moreover, the level of immunosuppressive cytokines, such as TGF-ß and IL-10, was significantly reduced in tumor microenvironment. DC vaccination in combination with lenalidomide plus PD-1 blockade has synergistically induced a strong antitumor immunity by modulating tumor microenvironment in a murine myeloma model. This protocol will become a promising translational approach to improve the efficacy of immunotherapy in the field of MM. DisclosuresNo relevant conflicts of interest to declare.

Chimeric Antigen Receptor 4SCAR-GD2-Modified T Cells Targeting High-Risk and Recurrent Neuroblastoma: A Phase II Multi-Center Trial in China

Background: Neuroblastoma (NB) is one of the most common childhood malignancies responsible for 15% of pediatric cancer deaths. 70% of children with NB present with high-risk features characterized by severe metastasis and poor long-term survival even if the multimodal intensive combination therapies are used. There is ongoing challenge for developing novel therapeutic strategies for NB. GD2 is a well-characterized tumor antigen in NB. Anti-GD2 antibodies and T cells genetically modified to express GD2-specific chimeric antigen receptor (GD2-CART) have demonstrated anti-cancer activity. Here we report the safety and efficacy of a new generation of GD2-CAR (4SCAR-GD2) targeting high-risk NB with a 1-year follow-up (NCI registrations: NCT02992210 and NCT02765243).Patients and Methods: Total 34 high-risk NB patients, after confirmation for GD2 expression, are enrolled from 5 hospitals in China. These patients had undergone a series of standard therapies, including surgery, chemo- and radio-therapy before CART treatment. CD3 positive cells were selected from apheresis lymphocytes and activated before lentiviral infection. The cells were transduced with a caspase 9-inducible, safety-engineered lentivector CAR containing multiple intracellular signaling domains: GD2-scFv/CD28/CD27/CD3ζ-iCasp9. Before CART infusion, patients received cyclophosphamide and fludarabine conditioning. The CART infusion dose was at average 1.1x10^6/kg. Toxicity was determined based on NCI common toxicity criteria. The International Neuroblastoma Response Criteria were used to define response after CART treatment. The total observation time for all patients is 1 year after infusion.Results: GD2 expression was scored 0 to 4 by immunohistochemical staining of tumor sections, and total 142 specimens were stained. The results showed that 9% of specimens were negative and more than 71% scored more than 2. Total 34 patients were infused with 4SCAR-GD2 T cells: 24% of them experienced no adverse reactions (per NCI-CTC v.4.0), 50% had grade 1 and 26% had grade 2 toxicity responses including fever, skin rash and peripheral nerve pain. No grade 3 or 4 toxicity was observed. CART expansion kinetics were monitored by quantitative PCR of CAR in peripheral blood. The 4SCAR-GD2 illustrated long term persistence with the highest level of copy number detected at 30% on day 180 after CART infusion. Marked tumor regression was observed in 2 patients. At 28 days after infusion, by ultrasound the mediastinal and peritoneal tumors in one patient were reduced by 94% and 99.5%, respectively. The other patient had progressive tumor located in retroperitoneum and PET-CT showed shrinking tumor two months after CART treatment; retroperitoneal SUV decreased from 2.5 to 1.5. At the one-year observation point, 38% of patients were in stable disease, 15% had partial response and 47% were in disease progression. The 1-year overall survival rate is 74%.Conclusion: This multicenter trial demonstrates that the 4SCAR-GD2 T cells are safe and effective for treating high-risk stage IV NB children. The correlations of GD2 expression in tumor, CART kinetics in blood and treatment responses could not be statistically established, suggesting a vast disease heterogeneity. Important therapeutic correlates require a large patient cohort and long-term follow-ups to elucidate. DisclosuresYu:H3 Biomedicine Inc.: Employment.

Antiangiogenesis Combined with Inhibition of the Hypoxia Pathway Facilitates Low-Dose, X-ray-Induced Photodynamic Therapy.

X-ray-induced photodynamic therapy (XPDT) is overwhelmingly superior in treating deep-seated cancers. However, limitations remain, owing to a combination of the poor scintillation performance of the nanoscintillator, low energy transfer efficiency of the therapeutic nanoplatform, and hypoxic environment presented in the tumor tissue. Collectively, these reduce the curative effect of XPDT. Here, we report a highly efficient, low-dose XPDT realized by systematic optimization from scintillation efficiency, nanoplatform structure, to therapeutic approach. We developed a biocompatible, codoped CaF2 nanoscintillator that emitted sufficiently green radioluminescence that was bright enough to be seen by the naked eye. Using dendrimers as a framework, we built a nanoplatform featuring a dual-core-satellite architecture, which enabled both procedurally and spatially separate dual-loading of therapeutic agents. This strategy allowed for the fabrication of a combined XPDT and antiangiogenic therapy, resulting in a therapeutic system capable of simultaneous tumor attacks. After exposure to ultralow dose radiation, XPDT resulted in marked tumor reduction while the antiangiogenic drug effectively blocked tumor vascularization exacerbated by XPDT-mediated hypoxia, rendering a pronounced synergy effect. This system also showed high biosafety, as the agents adopted had been used clinically and both Ca and F elements were widespread in the human body. Taken together, the findings presented here provided a reference for the construction of complex, multiloading architecture in coordination with structural complexity and functional diversification. This work provided a safer and more robust application of the combined XPDT and antiangiogenesis in future clinical treatment settings.

Combined Targeting of the Proteasome and Bcl-2 Sensitizes Human B-Cell Lymphoma and Multiple Myeloma to Cyclophosphamide in In Vitro and In Vivo Models of These Diseases.

The balance of apoptotic proteins is a critical mechanism used by the cell to maintain the mitochondrial transmembrane potential, which plays a critical role in governing apoptosis. Bortezomib (B) is a proteasome inhibitor that selectively inhibits the 26S proteasome, leading to an increase in many intracellular proteins, including proteins that lead to inhibition of NF-kB, as well as proapoptotic proteins like bik and Bax. Oblimersen (Obl) is a 18 mer oligonucleotide results in decreased transcription of the antiapoptotic protein bcl-2. We hypothesized that the mitochondrial membrane potential could be influenced by the co-administration of these agents to favor aopotosis. We report results of pre-clinical in vitro and in vivo experiments using these agents in combination in various lymphoma (NHL) & myeloma (MM) models. NHL & MM cell lines were exposed to Obl in the presence of cationic lipid and incubated for 48 hours. DNA synthesis & cell growth arrest was quantified by 3H-thymidine incorporation assays at 72 hrs, & revealed a concentration dependent cell killing. Induction of apoptosis in the presence or absence of different concentrations of B was detected by multiparameter flow cytometric analysis. Data reveals addition of bortezomib, even at relatively low concentrations (10 nM), additively improved the effects of Obl in all the cell lines studied. A SCID beige model of aggressive large B-cell lymphoma was used by injecting 1x10e7 cells (SKI-DLCL-1 or RL, a cell line over expressing bcl-2 through the 14:18 translocation) as a subcutaneous flank tumor. In over 6 in vivo experiments, the combination of B (0.5 mg/kg days 3 & 7) + Obl (3 mg/kg days 1,3,5, &7) consistently resulted in about 50% inhibition of growth of both SKI-DLCL-1 & RL compared to that in the control. Surprisingly, results were evident even using a low dose schedule of bortezomib (about one quarter of the MTD), suggesting the possibility of a small sensitizing dose of this drug. In all experiments, growth delay seen with the doublet exceeded the results noted with any singlet (B or Obl alone). Integration of cyclophosphamide at a dose of 50 mg/kg produced marked tumor shrinkage that appeared synergistic in several circumstances. Single agent CTX produced excellent growth delay (similar to any doublet), but in all these cases, tumor re-growth was evident by 21–25 days post tumor transplant. CTX dose & schedule was also important. Same day ‘lump and dump' of all drugs failed to effect cured, while CTX preceding B consistently produced best results, with cures found only in this triplet cohort. Results suggest that optimal scheduling of agents requires a ‘lead in' exposure of Obl, followed by CTX preceding all doses of bortezomib. These studies support the efficacy of this regimen.

Remodeling of the tumor microenvironment using an engineered oncolytic vaccinia virus improves PD-L1 inhibition outcomes.

Immune checkpoint inhibitor (ICI) immunotherapies have vastly improved therapeutic outcomes for patients with certain cancer types, but these responses only manifest in a small percentage of all cancer patients. The goal of the present study was to improve checkpoint therapy efficacy by utilizing an engineered vaccinia virus to improve the trafficking of lymphocytes to the tumor, given that such lymphocyte trafficking is positively correlated with patient checkpoint inhibitor response rates. We developed an oncolytic vaccinia virus (OVV) platform expressing manganese superoxide dismutase (MnSOD) for use as both a monotherapy and together with anti-PD-L1. Intratumoral OVV-MnSOD injection in immunocompetent mice resulted in inflammation within poorly immunogenic tumors, thereby facilitating marked tumor regression. OVV-MnSOD administration together with anti-PD-L1 further improved antitumor therapy outcomes in models in which these monotherapy approaches were ineffective. Overall, our results emphasize the value of further studying these therapeutic approaches in patients with minimally or non-inflammatory tumors.

EMBR-30. A NOVEL PLK1 INHIBITOR ONVANSERTIB EFFECTIVELY SENSITIZES GROUP 3 MEDULLOBLASTOMA TO RADIOTHERAPY

Abstract Medulloblastoma (MB) is often accompanied by MYC amplification. PLK1 is an oncogenic kinase that controls cell cycle and proliferation, and it has been preclinically validated as a cancer therapeutic target. Onvansertib (PCM-075) is a novel, orally available PLK1 inhibitor, which shows tumor growth inhibition in many types of cancer. We examined the effect of Onvansertib on MYC-driven medulloblastoma as a monotherapy or in combination with radiation. A Crisper-Cas9 screen was used to discover essential genes for MB tumor growth. Microarray and immunohistochemistry on pediatric patient samples were performed to examine the expression of PLK1. The effect of Onvansertib in vitro was measure by cell viability, colony-forming assays, extreme limiting dilution assay, and RNA-Seq. ALDH activity, cell-cycle distribution, and apoptosis were analyzed by flow cytometry. DNA damage was assessed by immunofluorescence staining. Medulloblastoma xenografts were generated to explore the monotherapy or radio-sensitizing effect. PLK1 is overexpressed in Group 3 MB. The IC50 concentrations of Onvansertib in Group 3 MB cell lines were between 4.9 and 6 nM. Onvansertib reduced colony formation, cell proliferation, stem cell renewal, and induced G2/M arrest in vitro. Moreover, Onvansertib in combination with radiation increased DNA damage and apoptosis compared with radiation alone. The combination of Onvansertib with radiotherapy resulted in marked tumor regression in orthotopic xenografts. These findings suggest that Onvansertib is an effective strategy in combination with radiotherapy in MB.

The synergy of BET inhibitors with aurora A kinase inhibitors in MYCN-amplified neuroblastoma is heightened with functional TP53

Amplification of MYCN is a poor prognostic feature in neuroblastoma (NBL) indicating aggressive disease. We and others have shown BET bromodomain inhibitors (BETi) target MYCN indirectly by downregulating its transcription. Here we sought to identify agents that synergize with BETi and to identify biomarkers of resistance. We previously performed a viability screen of ∼1,900 oncology-focused compounds combined with BET bromodomain inhibitors against MYCN-amplified NBL cell lines. Reanalysis of our screening results prominently identified inhibitors of aurora kinase A (AURKAi) to be highly synergistic with BETi. We confirmed the anti-proliferative effects of several BETi+AURKAi combinations in MYCN-amplified NBL cell lines. Compared to single agents, these combinations cooperated to decrease levels of N-myc. We treated both TP53-wild type and mutant, MYCN-amplified cell lines with the BETi JQ1 and the AURKAi Alisertib. The combination had improved efficacy in the TP53-WT context, notably driving apoptosis in both genetic backgrounds. JQ1+Alisertib combination treatment of a MYCN-amplified, TP53-null or TP53-restored genetically engineered mouse model of NBL prolonged survival better than either single agent. This was most profound with TP53 restored, with marked tumor shrinkage and apoptosis induction in response to combination JQ1+Alisertib. BETi+AURKAi in MYCN-amplified NBL, particularly in the context of functional TP53, provided anti-tumor benefits in preclinical models. This combination should be studied more closely in a pediatric clinical trial.

Phase I study of adoptive immunotherapy for advanced MUC1* positive breast cancer with autologous T cells engineered to express a chimeric antigen receptor, huMNC2-CAR44 specific for a cleaved form of MUC1 (MUC1*).

TPS2663 Background: Chimeric antigen receptor (CAR) T cell therapy targeting CD19 results in marked tumor regression for patients with CD19+ malignancies. It would be ideal to extend the success of CAR-T cell therapy to epithelial cancers. MUC1* is a post-translationally modified/cleaved form of mucin 1 (MUC1) that is frequently expressed on breast tumors, functions as a growth factor receptor, and a promising antigen for CAR-T cell therapy. Minerva Biotechnologies developed a CAR (huMNC2-CAR44) which recognizes MUC1* and does not bind to full-length or MUC1* negative cells. huMNC2-CAR44 product consists of autologous T cells transduced with a lentiviral vector encoding humanized MNC2-scFv (MUC1* targeting head), sequences from CD8 𝛼 leader, hinge and transmembrane domains, 4-1BB and CD3ζ domains. Methods: NCT04020575 is a phase I study evaluating the safety of adoptively transferred autologous T cells genetically modified to express huMNC2-CAR44 in patients with metastatic MUC1* positive breast cancer. After screening, leukapheresis is performed, CD8+ and CD4+ T cells are selected, transduced with huMNC2-CAR44, expanded, and antigen stimulated in vitro. Lymphodepletion with cyclophosphamide and fludarabine is followed by infusion of huMNC2-CAR44 CAR-T cells in escalating doses (3.3 x 105 CAR+ T cells/kg – 1 x 107 CAR+ T cells/kg). Key inclusion criteria include metastatic breast cancer of known ER, PR and HER2 status, MUC1* membrane expression > or = 30% with 2+ staining by IHC, measurable or evaluable disease, receipt of standard systemic therapies known to confer benefit, age > 18, informed consent, adequate organ function, and KPS > or = 60%. Patients with active autoimmune disease, uncontrolled infection, anticipated survival < 3 months, and/or untreated CNS metastases are not eligible. The primary objective is to identify the maximum tolerated (MTD) dose of huMNC2-CAR44 T cells by CTCAE v5 and Lee criteria. Secondary objectives include persistence and phenotype of adoptively transferred huMNC2-CAR44 T cells and preliminary antitumor activity. Exploratory objectives include trafficking of huMNC2-CAR44 T cells to tumor sites, effector function of huMNC2-CAR44 T cells in vivo, association between tumor MUC1* expression and huMNC2-CAR44 T cell persistence and response, change in tumor immune microenvironment by multiplex IHC in pre- and post-treatment tumor biopsies. Dose escalation is completed using a "3+3" design. Once the MTD has been determined, up to 15 more patients will be enrolled in each of 3 expansion cohorts (Luminal, HER2 positive, and TNBC) to inform future huMNC2-CAR44 T cell trials. Study is open to screening and enrollment in dose escalation. Up to 69 patients may be enrolled in dose escalation and expansion phases. Clinical trial information: NCT04020575.

Longitudinal single-cell profiling reveals molecular heterogeneity and tumor-immune evolution in refractory mantle cell lymphoma

The mechanisms driving therapeutic resistance and poor outcomes of mantle cell lymphoma (MCL) are incompletely understood. We characterize the cellular and molecular heterogeneity within and across patients and delineate the dynamic evolution of tumor and immune cell compartments at single cell resolution in longitudinal specimens from ibrutinib-sensitive patients and non-responders. Temporal activation of multiple cancer hallmark pathways and acquisition of 17q are observed in a refractory MCL. Multi-platform validation is performed at genomic and cellular levels in PDX models and larger patient cohorts. We demonstrate that due to 17q gain, BIRC5/survivin expression is upregulated in resistant MCL tumor cells and targeting BIRC5 results in marked tumor inhibition in preclinical models. In addition, we discover notable differences in the tumor microenvironment including progressive dampening of CD8+ T cells and aberrant cell-to-cell communication networks in refractory MCLs. This study reveals diverse and dynamic tumor and immune programs underlying therapy resistance in MCL.

Efficient delivery of oncolytic enterovirus by carrier cell line NK-92

Many members of the enterovirus family are considered as promising oncolytic agents; however, their systemic administration is largely inefficient due to the rapid neutralization of the virus in the circulation and the barrier functions of the endothelium. We aimed to evaluate natural killer cells as carriers for the delivery of oncolytic enteroviruses, which would combine the effects of cell immunotherapy with virotherapy. We tested four strains of nonpathogenic enteroviruses against the glioblastoma cell line panel and evaluated the produced infectious titers. Next, we explored whether these virus strains could be delivered to the tumor by natural killer cell line NK-92, which is being actively evaluated as a clinically acceptable therapeutic. Several strains of enteroviruses demonstrated oncolytic properties, but only coxsackievirus A7 (CVA7) could replicate in NK-92 cells efficiently. We compared the delivery efficiency of CVA7 in vivo, using NK-92 cells and direct intravenous administration, and found significant advantages of cell delivery even after a single injection. This suggests that the NK-92 cell line can be utilized as a vehicle for the delivery of the oncolytic strain of CVA7, which would improve the clinical potential of this viral oncolytic for the treatment of glioblastoma multiforme and other forms of cancer.

The KRASG12C Inhibitor MRTX849 Reconditions the Tumor Immune Microenvironment and Sensitizes Tumors to Checkpoint Inhibitor Therapy.

KRASG12C inhibitors, including MRTX849, are promising treatment options for KRAS-mutant non-small cell lung cancer (NSCLC). PD-1 inhibitors are approved in NSCLC; however, strategies to enhance checkpoint inhibitor therapy (CIT) are needed. KRASG12C mutations are smoking-associated transversion mutations associated with high tumor mutation burden, PD-L1 positivity, and an immunosuppressive tumor microenvironment. To evaluate the potential of MRTX849 to augment CIT, its impact on immune signaling and response to CIT was evaluated. In human tumor xenograft models, MRTX849 increased MHC class I protein expression and decreased RNA and/or plasma protein levels of immunosuppressive factors. In a KrasG12C -mutant CT26 syngeneic mouse model, MRTX849 decreased intratumoral myeloid-derived suppressor cells and increased M1-polarized macrophages, dendritic cells, CD4+, and CD8+ T cells. Similar results were observed in lung KrasG12C -mutant syngeneic and a genetically engineered mouse (GEM) model. In the CT26 KrasG12C model, MRTX849 demonstrated marked tumor regression when tumors were established in immune-competent BALB/c mice; however, the effect was diminished when tumors were grown in T-cell-deficient nu/nu mice. Tumors progressed following anti-PD-1 or MRTX849 single-agent treatment in immune-competent mice; however, combination treatment demonstrated durable, complete responses (CRs). Tumors did not reestablish in the same mice that exhibited durable CRs when rechallenged with tumor cell inoculum, demonstrating these mice developed adaptive antitumor immunity. In a GEM model, treatment with MRTX849 plus anti-PD-1 led to increased progression-free survival compared with either single agent alone. These data demonstrate KRAS inhibition reverses an immunosuppressive tumor microenvironment and sensitizes tumors to CIT through multiple mechanisms.

Macrophages Mediate the Antitumor Effects of the Oncolytic Virus HSV1716 in Mammary Tumors.

Oncolytic viruses (OV) have been shown to activate the antitumor functions of specific immune cells like T cells. Here, we show OV can also reprogram tumor-associated macrophage (TAM) to a less immunosuppressive phenotype. Syngeneic, immunocompetent mouse models of primary breast cancer were established using PyMT-TS1, 4T1, and E0771 cell lines, and a metastatic model of breast cancer was established using the 4T1 cell line. Tumor growth and overall survival was assessed following intravenous administration of the OV, HSV1716 (a modified herpes simplex virus). Infiltration and function of various immune effector cells was assessed by NanoString, flow cytometry of dispersed tumors, and immunofluorescence analysis of tumor sections. HSV1716 administration led to marked tumor shrinkage in primary mammary tumors and a decrease in metastases. This was associated with a significant increase in the recruitment/activation of cytotoxic T cells, a reduction in the presence of regulatory T cells and the reprograming of TAMs towards a pro-inflammatory, less immunosuppressive phenotype. These findings were supported by in vitro data demonstrating that human monocyte-derived macrophages host HSV1716 replication, and that this led to immunogenic macrophage lysis. These events were dependent on macrophage expression of proliferating cell nuclear antigen (PCNA). Finally, the antitumor effect of OV was markedly diminished when TAMs were depleted using clodronate liposomes. Together, our results show that TAMs play an essential role in support of the tumoricidal effect of the OV, HSV1716-they both host viral replication via a novel, PCNA-dependent mechanism and are reprogramed to express a less immunosuppressive phenotype.

MEK Inhibition Remodels the Immune Landscape of Mutant KRAS Tumors to Overcome Resistance to PARP and Immune Checkpoint Inhibitors.

Mutant KRAS tumors are associated with poor outcomes, at least in part, due to decreased therapeutic sensitivity. Here, we show that KRAS mutations are associated with resistance to monotherapy and combination therapy with PARP inhibitors (PARPi) and immune checkpoint blockade with anti-PD-L1 antibodies. In mutant KRAS tumors, inhibition of KRAS signaling with MEK inhibitors (MEKi) triggered and amplified PARPi-induced DNA damage, cytosolic double-stranded DNA accumulation, STING pathway activation, and CD8+ T-cell recruitment. Moreover, MEKi decreased myeloid-derived suppressor cell infiltration, in part, by inhibiting IL6 and GMCSF production. Importantly, addition of MEKi to PARPi and anti-PD-L1 resulted in marked tumor inhibition in immunocompetent mutant KRAS tumor models. This study provides the underlying mechanistic data to support evaluation of PARPi, MEKi, and anti-PD-L1 combination in clinical trials of mutant KRAS tumors. SIGNIFICANCE: This study provides key insights into the potential for using MEKi combined with PARPi and anti-PD-L1 for the treatment of all mutant KRAS tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/10/2714/F1.large.jpg.

Abstract OT-13-07: Phase I study of adoptive immunotherapy for advanced MUC1* positive breast cancer with autologous T cells engineered to express a chimeric antigen receptor, huMNC2-CAR44 specific for a cleaved form of MUC1 (MUC1*)

Abstract Background: Chimeric antigen receptor (CAR) T cell therapy targeting CD19 results in marked tumor regression for patients with B-cell malignancies. It would be ideal to extend the success of CAR T cell therapy to common epithelial cancers. MUC1* is a post-translationally modified/cleaved form of mucin 1 (MUC1) that is frequently expressed on breast tumors, functions as a growth factor receptor, and is a promising antigen for CAR T cell therapy. Minerva Biotechnologies developed a CAR T (huMNC2-CAR44) that specifically recognizes the cleaved form of MUC1* and does not bind to full-length or MUC1* negative cells. huMNC2-CAR44 product consists of autologous T cells transduced with a lentiviral vector encoding humanized MNC2-scFv (MUC1* targeting head), sequences from CD8 α;; leader, hinge and transmembrane domains, 4-1BB and CD3ζ domains. Trial Design: NCT04020575 is a phase I study evaluating the safety and anti-tumor activity of adoptively transferred autologous T cells genetically modified to express huMNC2-CAR44 in patients with metastatic MUC1* breast cancer. After screening, leukapheresis is performed, CD8+ and CD4+ T cells are selected, transduced with huMNC2-CAR44, expanded, and antigen stimulated in vitro. Lymphodepletion with cyclophosphamide and fludarabine is followed 36-96 hours later by infusion of huMNC2-CAR44 CAR T cells in escalating doses (3.3 x 105 CAR+ T cells/kg - 1 x 107 CAR+ T cells/kg). Eligibility: Key inclusion criteria include metastatic breast cancer of known ER, PR and HER2 status which has MUC1* membrane expression > or = 30% by immunohistochemistry, measurable or evaluable disease, receipt of standard systemic therapies known to confer benefit, age >18, informed consent, adequate organ function, and KPS > or = 60%. Patients with active autoimmune disease or uncontrolled infection, contraindication to cyclophosphamide, anticipated survival < 3 months, and/or untreated CNS metastases are not eligible. Specific Aims: The primary objective is to identify the maximum tolerated dose of huMNC2-CAR44 T cells by CTCAE v5 and Lee criteria. Secondary objectives include persistence and phenotype of adoptively transferred huMNC2-CAR44 T cells and preliminary antitumor activity in all patients with measurable disease by RECIST 1.1. Exploratory objectives include trafficking of huMNC2-CAR44 T cells to tumor sites, effector function of huMNC2-CAR44 T cells in vivo, association between tumor MUC1* expression and huMNC2-CAR44 T cell persistence and response, change in tumor immune microenvironment by multiplex immunohistochemistry in pre and post-treatment tumor biopsies. Statistical Design: Dose escalation or de-escalation is tested in cohorts of 3 patients each using standard "3+3" dose-finding targeting a T cell dose that is associated with a true DLT rate < 33% and > than 17%. DLT period is between day 0 and 28. Once the MTD has been determined, up to 15 more patients will be enrolled in each of 3 expansion cohorts (Luminal, HER2 positive, and TNBC) to estimate the anti-tumor activity in these patient populations and to inform future huMNC2-CAR44 T cell trials. Present accrual: Study is open to screening and enrollment in dose escalation. Up to 69 patients may be enrolled in dose escalation and expansion phases. Contact information: To refer patients or obtain more information, please contact immunotherapy@seattlecca.org. Citation Format: Jennifer Marie Specht, David Maloney, Cecilia Yeung, Qian (Vicky) Wu, Cynthia Bamdad. Phase I study of adoptive immunotherapy for advanced MUC1* positive breast cancer with autologous T cells engineered to express a chimeric antigen receptor, huMNC2-CAR44 specific for a cleaved form of MUC1 (MUC1*) [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 OT-13-07.