Jan ter Meulen

Virological and preclinical characterization of a dendritic cell targeting, integration-deficient lentiviral vector for cancer immunotherapy.

Dendritic cells (DCs) are essential antigen-presenting cells for the initiation of cytotoxic T-cell responses and therefore attractive targets for cancer immunotherapy. We have developed an integration-deficient lentiviral vector termed ID-VP02 that is designed to deliver antigen-encoding nucleic acids selectively to human DCs in vivo. ID-VP02 utilizes a genetically and glycobiologically engineered Sindbis virus glycoprotein to target human DCs through the C-type lectin DC-SIGN (CD209) and also binds to the homologue murine receptor SIGNR1. Specificity of ID-VP02 for antigen-presenting cells in the mouse was confirmed through biodistribution studies showing that following subcutaneous administration, transgene expression was only detectable at the injection site and the draining lymph node. A single immunization with ID-VP02 induced a high level of antigen-specific, polyfunctional effector and memory CD8 T-cell responses that fully protected against vaccinia virus challenge. Upon homologous readministration, ID-VP02 induced a level of high-quality secondary effector and memory cells characterized by stable polyfunctionality and expression of IL-7R&agr;. Importantly, a single injection of ID-VP02 also induced robust cytotoxic responses against an endogenous rejection antigen of CT26 colon carcinoma cells and conferred both prophylactic and therapeutic antitumor efficacy. ID-VP02 is the first lentiviral vector which combines integration deficiency with DC targeting and is currently being investigated in a phase I trial in cancer patients.

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.

First-in-Human Treatment With a Dendritic Cell-targeting Lentiviral Vector-expressing NY-ESO-1, LV305, Induces Deep, Durable Response in Refractory Metastatic Synovial Sarcoma Patient

Effective induction of antitumor T cells is a pivotal goal of cancer immunotherapy. To this end, lentiviral vectors (LV) are uniquely poised to directly prime CD8 T-cell responses via transduction of dendritic cells in vivo and have shown promise as active cancer therapeutics in preclinical tumor models. However, until now, significant barriers related to production and regulation have prevented their widespread use in the clinic. We developed LV305, a dendritic cell-targeting, integration-deficient, replication incompetent LV from the ZVex platform, encoding the full-length cancer-testis antigen NY-ESO-1. LV305 is currently being evaluated in phase 1 and 2 trials in metastatic recurrent cancer patients with NY-ESO-1 positive solid tumors as a single agent and in combination with anti-PD-L1. Here we report on the first patient treated with LV305, a young woman with metastatic, recurrent, therapy-refractive NY-ESO-1+ synovial sarcoma. The patient developed a robust NY-ESO-1-specific CD4+ and CD8+ T-cell response after 3 intradermal injections with LV305, and subsequently over 85% disease regression that is continuing for >2.5 years posttherapy. No adverse events >grade 2 occurred. This case demonstrates that LV305 can be safely administered and has the potential to induce a significant clinical benefit and immunologic response in a patient with advanced stage cancer.Effective induction of antitumor T cells is a pivotal goal of cancer immunotherapy. To this end, lentiviral vectors (LV) are uniquely poised to directly prime CD8 T-cell responses via transduction of dendritic cells in vivo and have shown promise as active cancer therapeutics in preclinical tumor models. However, until now, significant barriers related to production and regulation have prevented their widespread use in the clinic. We developed LV305, a dendritic cell-targeting, integration-deficient, replication incompetent LV from the ZVex platform, encoding the full-length cancer-testis antigen NY-ESO-1. LV305 is currently being evaluated in phase 1 and 2 trials in metastatic recurrent cancer patients with NY-ESO-1 positive solid tumors as a single agent and in combination with anti-PD-L1. Here we report on the first patient treated with LV305, a young woman with metastatic, recurrent, therapy-refractive NY-ESO-1 synovial sarcoma. The patient developed a robust NY-ESO-1-specific CD4 and CD8 T-cell response after 3 intradermal injections with LV305, and subsequently over 85% disease regression that is continuing for >2.5 years posttherapy. No adverse events >grade 2 occurred. This case demonstrates that LV305 can be safely administered and has the potential to induce a significant clinical benefit and immunologic response in a patient with advanced stage cancer.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0/.

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.

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

Abstract 5092: ZVex® lentiviral vector strongly activates pro-inflammatory, antigen processing, and anti-viral defense response pathways in monocyte-derived dendritic cells

Background: Dendritic cells (DCs) are professional antigen presenting cells that effectively bridge the innate and adaptive immune responses and require activation for successful priming of naive T-cells. We have developed ZVex, an integration deficient, hybrid lentiviral vector engineered to target DC-SIGN expressed on immature myeloid DCs for genetic immunization against tumor antigens. As lentiviruses normally don9t efficiently infect DCs, little is known about the functional effect of LV transduction of conventional DCs. Here, the effect of DC transduction with ZVex vectors was studied by gene expression profiling. Material and Methods: Human moDCs from multiple donors were prepared by 5-day stimulation with IL-4 and GM-CSF and transduced with ZVex-GFP or control vectors, such as a ZVex with nonfunctional reverse transcriptase (RT-dead), ZVex particles generated to contain no vector genome (empty ZVex), and a heat-inactivated ZVex preparation. Cellular RNA was isolated at different time points from transduced DC cultures and used for gene expression profiling with Nanostring’s human pan cancer immune panel (770 genes). Results: DCs transduced with ZVex vectors displayed statistically significant up-regulation of genes involved in pro-inflammatory, antigen processing, and anti-viral defense pathways. Noteworthy among the up-regulated genes were classical anti-viral response genes like OAS3, MX1, and interferon stimulated genes like ISG15 and ISG20. Incubation with the RT-dead mutant vector also led to up-regulation of these genes, albeit to a much lower extent, suggesting that LV-RNA itself can result in the activation of these pathways in DCs, though reverse transcription appears to further potentiate the response. Of note, cells transduced with empty ZVex, which consists of an intact virion particle but lacks encapsulated LV RNA, also mediated a low magnitude and breadth of DC transcriptional activation, possibly via signaling through DC-SIGN. Conclusions: ZVex incorporates several elements capable of inducing a potent innate immune activation in DCs. DCs are relatively insensitive to lentiviral infection, but the use of accessory proteins in ZVex makes DCs conducive to ZVex transduction and results in the induction of a Type-1 IFN response that seems to be largely dependent on reverse transcription. In addition, other engineered vector particle components such as non-reverse transcribed viral RNA, viral structural proteins, and/or engagement of the DC-SIGN receptor, also contribute to DC activation. Citation Format: Anshika Bajaj, Lisa Y. Ngo, Peter Berglund, Jan ter Meulen. ZVex® lentiviral vector strongly activates pro-inflammatory, antigen processing, and anti-viral defense response pathways in monocyte-derived dendritic cells [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 5092. doi:10.1158/1538-7445.AM2017-5092

Abstract 4885: Intratumoral injection of G100 (TLR4 agonist glycopyranosyl lipid A) modulates tumor microenvironment and induces CD8 T cell-dependent, systemic anti-tumor immunity

The tumor microenvironment (TME) plays a critical role in controlling the balance between tumor progression and immune surveillance. Increased infiltration of T cells, especially CD8 T cells has been associated with a good prognosis. We hypothesized that G100, a novel synthetic TLR4 agonist, can modulate TME when directly injected into the tumor and trigger both a local and systemic effective immune response. Balb/c mice with implanted syngeneic A20 lymphoma received intratumoral (IT) injection of G100 (10 ig) or control PBS three times a week. This treatment significantly inhibited tumor growth and resulted in complete tumor regression in approximately 60% of treated mice. To investigate the effects of G100 on TME, tumors were collected after three IT G100 injections for gene expression analysis by Nanostring and immune phenotyping analysis by FACS. Out of the 770 immune response genes included in the mouse PanCancer Immune Profiling panel, 295 genes were significantly upregulated in G100 treated tumors. The upregulated genes include DC function-related genes (CD40, CD83, CD86, and Ly96) and T cell and NK cell function genes and multiple chemokines and cytokines (IL1b, IL12A, IL18, IL6, IFNa, FcaR4, ICOS, GZMB, CCL3, CCL5, CCL7, CXCL1, CXCL2, CXCL11, CCR5, CCR6, and CCR7). T cell exhaustion markers (CTLA4 and LAG3) and CD274 (PD-L1) were also induced. G100-induced inflammation is also reflected at the cellular level as shown by increased T cells and NK cells in tumor via FACS analysis. To determine the immune cells that mediated tumor rejection, mice were selectively depleted of CD4 or CD8 T cells during G100 treatment. Results showed that the anti-tumor effect of G100 is dependent on CD8 T cells. G100-induced tumor protection was durable as mice surviving the first tumor challenge rejected a secondary tumor challenge without additional G100 treatment. Altogether, our results showed that IT G100 induces a proinflammatory cytokine and chemokine milieu that changes a “cold” tumor to a “hot” tumor, which facilitates the development of a CD8 T cell-dependent potent and durable anti-tumor effect. The induction of PD-L1 also suggests the potential synergy between G100 and checkpoint blockade therapy. These preclinical data support an on-going clinical trial of IT G100 in patients with follicular non-Hodgkin9s lymphoma (NCT02501473), alone and in combination with anti-PD-1 therapy (pembrolizumab). Citation Format: Hailing Lu, Jessica Hewitt, Jan ter Meulen. Intratumoral injection of G100 (TLR4 agonist glycopyranosyl lipid A) modulates tumor microenvironment and induces CD8 T cell-dependent, systemic anti-tumor immunity. [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 4885.

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.