Tina C. Albershardt

Multiple BH3 Mimetics Antagonize Antiapoptotic MCL1 Protein by Inducing the Endoplasmic Reticulum Stress Response and Up-regulating BH3-only Protein NOXA

BH3 mimetics are small molecules designed or discovered to mimic the binding of BH3-only proteins to the hydrophobic groove of antiapoptotic BCL2 proteins. The selectivity of these molecules for BCL2, BCL-XL, or MCL1 has been established in vitro; whether they inhibit these proteins in cells has not been rigorously investigated. In this study, we used a panel of leukemia cell lines to assess the ability of seven putative BH3 mimetics to inhibit antiapoptotic proteins in a cell-based system. We show that ABT-737 is the only BH3 mimetic that inhibits BCL2 as assessed by displacement of BAD and BIM from BCL2. The other six BH3 mimetics activate the endoplasmic reticulum stress response inducing ATF4, ATF3, and NOXA, which can then bind to and inhibit MCL1. In most cancer cells, inhibition of one antiapoptotic protein does not acutely induce apoptosis. However, by combining two BH3 mimetics, one that inhibits BCL2 and one that induces NOXA, apoptosis is induced within 6 h in a BAX/BAK-dependent manner. Because MCL1 is a major mechanism of resistance to ABT-737, these results suggest a novel strategy to overcome this resistance. Our findings highlight a novel signaling pathway through which many BH3 mimetics inhibit MCL1 and suggest the potential use of these agents as adjuvants in combination with various chemotherapy strategies.

Vinblastine Induces Acute, Cell Cycle Phase–Independent Apoptosis in Some Leukemias and Lymphomas and Can Induce Acute Apoptosis in Others when Mcl-1 Is Suppressed

Chemotherapeutic agents modify intracellular signaling that culminates in the inhibition of Bcl-2 family members and initiates apoptosis. Inhibition of the extracellular signal-regulated kinase by PD98059 dramatically accelerates vinblastine-mediated apoptosis in ML-1 leukemia with cells dying in 4 hours from all phases of the cell cycle. Inhibition of protein synthesis by cycloheximide also markedly accelerated vinblastine-induced apoptosis, showing that the proteins required for this acute apoptosis are constitutively expressed. Vinblastine induced the rapid induction of Mcl-1 that was inhibited by PD98059 and cycloheximide. No change in Bcl-2 or Bcl-X was observed. We hypothesize that ML-1 cells use Mcl-1 for protection from the rapid vinblastine-induced apoptosis. This was confirmed by targeting Mcl-1 with short hairpin RNA. We also investigated the response of 13 other leukemia and lymphoma cell lines and cells from seven chronic lymphocytic leukemia patients. Four cell lines and all chronic lymphocytic leukemia cells were killed in 6 hours by vinblastine alone. Two additional cell lines were sensitized to vinblastine by PD98059, which suppressed Mcl-1. This acute apoptosis either alone or in combination with PD98059 required vinblastine-mediated activation of c-Jun-NH2-terminal kinase. PD98059 did not suppress Mcl-1 in other cell lines whereas sorafenib did, but this did not sensitize the cells to vinblastine, suggesting that the acute apoptosis varies depending on which Bcl-2 protein mediates protection. Most of the cell lines were sensitized to vinblastine by cycloheximide, suggesting that inhibition of a short-lived protein in addition to Mcl-1 can acutely sensitize cells. These results suggest several clinical strategies that might provide an effective therapy for selected patients. Mol Cancer Ther; 9(4); 791–802. ©2010 AACR.

LV305, a dendritic cell-targeting integration-deficient ZVexTM-based lentiviral vector encoding NY-ESO-1, induces potent anti-tumor immune response

We have engineered an integration-deficient lentiviral vector, LV305, to deliver the tumor antigen NY-ESO-1 to human dendritic cells in vivo through pseudotyping with a modified Sindbis virus envelop protein. Mice immunized once with LV305 developed strong, dose-dependent, multifunctional, and cytotoxic NY-ESO-1-specific cluster of differentiation 8 (CD8) T cells within 14 days post-immunization and could be boosted with LV305 at least twice to recall peak-level CD8 T-cell responses. Immunization with LV305 protected mice against tumor growth in an NY-ESO-1-expressing CT26 lung metastasis model, with the protective effect abrogated upon depletion of CD8 T cells. Adoptive transfer of CD8 T cells, alone or together with CD4 T cells or natural killer cells, from LV305-immunized donor mice to tumor-bearing recipient mice conferred significant protection against metastatic tumor growth. Biodistribution of injected LV305 in mice was limited to the site of injection and the draining lymph node, and injected LV305 exhibited minimal excretion. Mice injected with LV305 developed little to no adverse effects, as evaluated by toxicology studies adherent to good laboratory practices. Taken together, these data support the development of LV305 as a clinical candidate for treatment against tumors expressing NY-ESO-1.

Abstract 5477: Multiple BH3 mimetics antagonize anti-apoptotic MCL1 by inducing the endoplasmic reticulum stress response and upregulating the BH3-only protein NOXA

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Many small molecules have been designed or discovered that mimic the binding of BH3-only proteins to the hydrophobic groove of anti-apoptotic BCL2 proteins and thus have potential as anticancer agents. For example, ABT-737 preferentially binds and inhibits BCL2 and BCL-XL but not MCL1. It is generally believed that specific inhibitors of anti-apoptotic BCL2 proteins should induce apoptosis in a BAX/BAK-dependent manner. While this is true for ABT-737, many other purported BH3 mimetics have been shown to kill cells in a BAX/BAK-independent manner leading to the conclusion that they function through alternative undefined targets. The selectivity of these BH3 mimetics for BCL2, BCL-XL, or MCL1 has been established in vitro; whether they inhibit these proteins in cells has not been rigorously investigated. In this study, we used a panel of leukemia cell lines to assess the ability of seven putative BH3 mimetics to affect the interaction of BCL2 family members in a cell-based system. We show that ABT-737 is the only BH3 mimetic that inhibits BCL2 in cells as assessed by displacement of BAD from BCL2. The other six BH3 mimetics activated the endoplasmic reticulum (ER) stress response and induced ATF3 resulting in induction of NOXA which binds to and inhibits the anti-apoptotic MCL1 protein. The fact that 6 different compounds, all of which are thought to be BH3 mimetics, can induce ER stress suggests that they may interact with a common target although whether this is a BCL2 family member remains to be established. In most cancer cells, inhibition of either BCL2/X or MCL1 does not acutely induce apoptosis. However, by combining two BH3 mimetics, one that inhibits BCL2 and one that induces NOXA, apoptosis is induced within 6 h in a BAX/BAK-dependent manner. As MCL1 is a major mechanism of resistance to ABT-737, these results suggest a novel strategy to overcome this resistance. In summary, these results identify a novel signaling pathway through which many BH3-mimetics inhibit MCL1 and suggest the potential use of these agents as adjuvants in combination with various chemotherapy strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5477. doi:10.1158/1538-7445.AM2011-5477

Abstract 4884: Intratumoral expression of IL-12 from a dendritic cell-targeting chimeric lentiviral vector from the ZVex platform cures established tumors in multiple models and induces systemic anti-tumor responses

Interleukin-12 (IL-12), produced by antigen-presenting cells, plays a pivotal role in the interplay between innate and adaptive arms of the immune system. IL-12 treatment has been shown to augment cytotoxic T lymphocyte (CTL) and T-helper 1 responses and anti-tumor effects. However, its use as a systemic therapeutic agent is limited due to toxicity. Intratumoral administration of IL-12 is thus being explored as a local alternative route of administration. Such strategies involve plasmid electroporation or other methods that randomly direct cells in the tumor to express IL-12. Here, we evaluated whether targeting expression of IL-12 to intratumoral dendritic cells (DC), thus mimicking the cytokine9s physiological biosynthesis and localization, would result in local and systemic immune responses and tumor control in preclinical models. Six murine tumor models were used, including melanoma (B16F10), colon carcinoma (CT26), breast cancer (4T1), lymphoma (A20) and mastocytoma (P815). Tumors were implanted unilaterally, and for some models also bilaterally, to study systemic (abscopal) effects of therapy. A chimeric third-generation lentiviral vector from the ZVex platform, pseudotyped with the DC-tropic envelope glycoprotein of Sindbis virus, was engineered to express murine IL-12 (p35-p40) (ZVex/IL-12). ZVex/IL-12 was administered as a single intratumoral injection into palpable tumors, alone or in combination with the synthetic TLR4-agonist, glucopyranosyl lipid A (GLA). In some models, systemic anti-CTLA4 treatment was added to enhance clinical efficacy. Animals were monitored 2-3 times per week for tumor size and survival. Greatest curative efficacy of ZVex/IL-12 was observed in the 100% lethal CT26 flank model, where all treated animals cleared their tumors and survived until end-of-study at 70 days post-challenge. In the B16 footpad and flank models, curative efficacy varied from 40% to 90%, with abscopal effects being observed after addition of anti-CTLA4. In the A20 and P815 models, efficacy varied from 30% to 60%. In the aggressive orthotopic 4T1 breast cancer model, co-administration of ZVex/IL-12 with GLA-AF resulted in significantly delayed tumor growth and increased survival time compared to either ZVex/IL-12 or GLA-AF used alone. A single intratumoral injection of ZVex/IL-12 resulted in complete tumor regression or significant growth delay in all mouse tumor models investigated and was accompanied by significant survival benefit. The therapeutic effect could be enhanced by ad-mixing with GLA. Abscopal effects were observed in some models after treatment with ZVex/IL12 only and in others after addition of anti-CTLA4. These results point to the powerful modification of the tumor/tumor microenvironment by intratumorally expressed IL-12 by a ZVex vector and the induction of local and systemic immunity. Citation Format: Tina C. Albershardt, Anshika Bajaj, Jacob F. Archer, Rebecca S. Reeves, Andrea J. Parsons, Lisa Y. Ngo, Jan ter Meulen, Peter Berglund. Intratumoral expression of IL-12 from a dendritic cell-targeting chimeric lentiviral vector from the ZVex platform cures established tumors in multiple models and induces systemic anti-tumor responses. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4884.

A "prime-pull" immunotherapy approach using a lentiviral vector and intratumoral TLR4 agonist redirects cytotoxic T cells

The clinical efficacy of tumor specific effector T cells is limited by their proper trafficking to the site of the tumor and the locally immunosuppressive environment. Strategies to improve homing and activity of immune effector cells to tumors are needed to unlock the potential of active cancer immunotherapy.

Abstract 5673: Large established B16 tumors in mice are eradicated by ZVex® (dendritic cell-targeting lentiviral vector) and G100 (TLR4 agonist) combination immunotherapy through increasing tumor-infiltrating effector T cells and inducing antigen spreading

INTRODUCTION: Effective immunotherapy requires the presence of effector T cells penetrating the tumor. ZVex is a hybrid lentiviral vector platform that targets dendritic cells in vivo to express genes of tumor-associated antigen (TAA)-of-interest and activate TAA-specific CD8 T cells. G100 is the intratumoral (IT) injection of formulated glucopyranosyl lipid A (a synthetic TLR4 agonist) and has been shown to induce T cell homing chemokines, CXCL9 and CXCL10. We report here that G100 promoted an inflamed tumor microenvironment (TME) and improved infiltration of ZVex-induced TAA-specific CD8 T cells to the TME, thereby eradicating large established B16 tumors. This was previously achieved only with a complex vaccine/anti-tumor antibody/checkpoint inhibitor/IL-2 regimen (Moynihan, Nature, 2016). RESULTS: B16-OVA tumor-bearing mice were randomized into 4 treatment cohorts: 1) untreated; 2) ZVex expressing ovalbumin (ZVex/OVA), subcutaneously (SC); 3) G100 (IT); 4) ZVex/OVA (SC) and G100 (IT) combination. While mice in Cohorts 2 and 3 exhibited delayed tumor growth, nearly all mice (16/18) from Cohort 4 had completely regressed tumors and survived tumor-free until end of study (109 days). CD8 T cell depletion abrogated this anti-tumor response. Cohort 4 mice with regressed tumors were then randomized and re-challenged with either a) B16-OVA or b) parental B16 (lacking expression of the ZVex-targeted antigen, OVA): a) B16-OVA re-challenge was rejected by 100% of recipient mice - consistent with induction of T cell memory; and b) B16 re-challenge was rejected by 30%-50% of recipient mice - demonstrating functional antigen spreading. T cell receptor deep sequencing showed that ZVex/OVA alone increased tumor-infiltrating T cell clones specific for OVA, validated by pMHC-multimer staining. ZVex/OVA and G100 combination expanded additional T cell clones, further evident of antigen spreading. Lastly, the TME of Cohort 4 mice showed the most profound pro-inflammatory changes, as assessed by RNA transcriptional profiling. CONCLUSIONS: These data collectively demonstrate that anti-tumor efficacy observed in Cohort 4 mice was mediated largely by ZVex/OVA-induced effector T cells and that TME modulation with G100 drastically shifted the TME to a more inflamed milieu, promoting T cell proliferation, antigen spreading, and generation of immunological memory. To our knowledge, this is the first time that large established B16 tumors have been completely eradicated using a combination of systemic and in situ immunizations, a potentially effective strategy to convert non-inflamed tumors to inflamed tumors. Both ZVex and G100 are in multiple clinical trials, and their combination is currently being investigated in a phase I trial in soft tissue sarcoma patients with injectable tumors. Citation Format: Tina C. Albershardt, Andrea J. Parsons, Jardin Leleux, Peter Berglund, Jan ter Meulen. Large established B16 tumors in mice are eradicated by ZVex® (dendritic cell-targeting lentiviral vector) and G100 (TLR4 agonist) combination immunotherapy through increasing tumor-infiltrating effector T cells and inducing antigen spreading [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5673. doi:10.1158/1538-7445.AM2017-5673

Checkpoint inhibitors synergize with therapeutic platforms, ZVex and GLAAS by enhancing lentiviral vector-induced tumor-specific immunity and adjuvant-mediated anti-tumor efficacy

The dysregulation of immune checkpoints by tumors is an important mechanism of immune resistance, as administration of checkpoint inhibitors has resulted in impressive clinical responses in patients with late stage cancers. However, a subset of patients exhibits insufficient or no clinical response presumably due to the absence of tumor-specific cytotoxic T lymphocytes (CTLs). This supports the rationale to combine checkpoint inhibitors with therapeutic platforms that generate effector T cells. We have previously shown that the lentiviral vector platform, ZVexTM, generates high levels of tumor antigen-specific CTLs that are critical in mediating protection against tumor challenge in mice. Protection was enhanced by subsequent intramuscular injections of recombinant tumor antigen protein with GLA-SE (formulated glucopyranosyl lipid A, a synthetic TLR4 agonist that is the central component of the GLAASTM platform) or intratumoral injections of G100 (GLA-SE without protein antigen). ZVex and GLAAS are thus complementary platforms capable of generating tumor-specific immunity through the in vivo induction of antigen-specific T cells. Here, we evaluated whether checkpoint blockade could further enhance anti-tumor immunity induced by ZVex and/or GLAAS in mouse tumor models. In C57BL/6 mice, anti-PD-1 or anti-PD-L1 – but not anti-CTLA4 – enhanced a ZVex/mTRP1-induced CD8 T cell response. To determine whether checkpoint inhibition impacted therapeutic efficacy, B16F10 tumor-bearing mice were immunized with ZVex/mTRP1 followed by weekly administrations of anti-PD-1, anti-PD-L1, or anti-CTLA4. Mirroring the immunogenicity results, ZVex/mTRP1-induced anti-tumor protection was enhanced by the addition of anti-PD-1 or anti-PD-L1, but not anti-CTLA4. This is consistent with previous studies in which we demonstrated that the magnitude of antigen-specific CTL response correlated with the degree of anti-tumor protection. Additionally, compared to either anti-PD-1 or anti-PD-L1 alone, the combined administration of both checkpoint inhibitors best improved anti-tumor efficacy induced by ZVex/mTRP1, or G100, or the combination of ZVex/mTRP1 and GLA-SE/TRP1 protein. As PD-L1 is not the only ligand for PD-1 and PD-1 is not the only receptor for PD-L1, these findings suggest that inhibition of signaling through either PD-1 or PD-L1 alone may not be sufficient to completely block the PD-1/PD-L1 checkpoint. Taken together, combining checkpoint inhibitors with ZVex and GLAAS capitalizes on the strength of each therapeutic platform. ZVex and GLAAS efficiently generate the effector T cells needed for an effective anti-tumor response. By blocking active immune checkpoints, checkpoint inhibitors further enhance ZVex- and/or GLAAS-induced anti-tumor immunity. Our findings support the combination of checkpoint inhibitors with ZVex and/or GLAAS in clinical trials.

Intratumoral injections of G100 (synthetic TLR4 agonist) increase trafficking of lentiviral vector-induced antigen-specific CD8 T cells to the tumor microenvironment.

The clinical efficacy of tumor-specific effector T cells can be limited by their proper trafficking to the site of the tumor and the immunosuppressed local environment. Strategies to improve homing of effector cells to tumors and to enhance activity of these effector cells could further unlock the potential of active cancer immunotherapy. G100 is the synthetic TLR4 agonist glucopyranosyl lipid adjuvant (GLA) formulated with an oil-in-water stable emulsion and has been shown to induce T cell homing chemokines CXCL9 and CXCL10. We assessed here whether intratumoral injections of G100 could improve trafficking of tumor antigen-specific CD8 T cells to the tumor microenvironment (TME), thereby achieving better anti-tumor control. Untreated, B16F10-OVA tumor-bearing mice generated no detectable levels of ovalbumin (OVA)-specific CD8 T cell response as assessed by flow cytometry analysis. In contrast, tumor-bearing mice immunized with ZVexTM/OVA, a novel lentiviral vector platform expressing OVA, generated 8-9% tumor antigen-specific effector and memory CD8 T cells within the peripheral tissue, which remained detectable at low levels even up to 35 days post-immunization. Tumor-infiltrating lymphocytes (TILs) isolated from mice treated with ZVex/OVA alone had an average of 16.6% antigen-specific CD8 T cells, whereas those from mice treated with ZVex/OVA and G100 had 25.9%. While ZVex/OVA-induced antigen-specific CD8 T cells infiltrated the tumor without G100, most of these CD8 T cells did not remain in the TME over time. Intratumoral injections of G100 not only increased the total number of effector CD8 T cells within the TME but also kept the CD8 T cells within the TME over time. Furthermore, tumor-bearing mice treated with ZVex/OVA and G100 had significantly improved survival with slower growing tumors. We show here that intratumoral injections of a formulated synthetic TLR4 agonist, G100, improved vector-induced therapeutic efficacy by increasing trafficking of vector-induced effector T cells toward the TME. Because G100 also stimulates antigen presentation and maturation of dendritic cells, intratumoral G100 following vector-induced generation of antigen-specific CD8 T cells or adoptive transfer of CAR or TCR T cells may be an effective way to increase the therapeutic efficacy of cancer immunotherapy.

Abstract 2817: DCVex(TM): A novel DC-targeted vector platform for cancer immunotherapy

Dendritic cells (DCs) are essential for the initiation of T cell responses and are therefore an attractive target for cancer immunotherapy. The DC vaccine, Provenge®, as well as a number of on-going clinical trials, have in principle validated this concept. However, the currently pursued strategy of ex vivo immunization of DCs is time-consuming and costly. We have developed a 3rd generation, integration-deficient lentivector platform, DCVex(TM), which is designed to deliver tumor antigen-encoding genes directly to DCs in vivo by targeting the DC-SIGN receptor. Mice immunized with DCVex(TM) vectors expressing a variety of model antigens developed strong, dose-dependent poly-functional and cytotoxic antigen-specific CD8 T cell responses, as assessed by intracellular cytokine staining and in vitro cytotoxic T lymphocyte assays. Repeated immunizations resulted in boosting of the T cell responses, indicating the absence of induction of inhibitory anti-vector immunity. Importantly, in stringent therapeutic tumor models (e.g., B16F10 footpad melanoma and CT26 lung metastasis models), immunization with tumor antigen-encoding vectors protected the majority of animals from death in a dose-dependent manner. These findings demonstrate the potential of DCVex(TM) as a novel cancer vaccine platform suitable for in vivo DC immunization. Citation Format: Tina C. Albershardt, Semih U. Tareen, Jared M. Odegard, David J. Campbell, Patrick Flynn, Scott H. Robbins, Peter Berglund, Jan H. ter Meulen. DCVex(TM): A novel DC-targeted vector platform for cancer immunotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2817. doi:10.1158/1538-7445.AM2014-2817