Peter Berglund

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.

HIV-1 Conserved Mosaics Delivered by Regimens with Integration-Deficient DC-Targeting Lentiviral Vector Induce Robust T Cells

To be effective against HIV type 1 (HIV-1), vaccine-induced T cells must selectively target epitopes, which are functionally conserved (present in the majority of currently circulating and reactivated HIV-1 strains) and, at the same time, beneficial (responses to which are associated with better clinical status and control of HIV-1 replication), and rapidly reach protective frequencies upon exposure to the virus. Heterologous prime-boost regimens using virally vectored vaccines are currently the most promising vaccine strategies; nevertheless, induction of robust long-term memory remains challenging. To this end, lentiviral vectors induce high frequencies of memory cells due to their low-inflammatory nature, while typically inducing only low anti-vector immune responses. Here, we describe construction of novel candidate vaccines ZVex.tHIVconsv1 and ZVex.tHIVconsv2, which are based on an integration-deficient lentiviral vector platform with preferential transduction of human dendritic cells and express a bivalent mosaic of conserved-region T cell immunogens with a high global HIV-1 match. Each of the two mosaic vaccines was individually immunogenic. When administered together in heterologous prime-boost regimens with chimpanzee adenovirus and/or poxvirus modified vaccinia virus Ankara (MVA) vaccines to BALB/c and outbred CD1-Swiss mice, they induced a median frequency of over 6,000 T cells/106 splenocytes, which were plurifunctional, broadly specific, and cross-reactive. These results support further development of this vaccine concept.

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

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

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 2505: Therapeutic efficacy of the ZVex™ and GLAAS™ platforms in a B16-F10/hCAIX melanoma mouse model

ZVex™ and GLAAS™ are two dendritic-cell (DC) targeting platform technologies designed to enhance immune responses through the in vivo induction of antigen specific CD8 and CD4 T-cells, respectively. ZVex™ is a lentiviral vector pseudotyped with a modified Sindbis virus envelope engineered to deliver tumor antigen-encoding nucleic acids to dendritic cells in vivo. GLAAS™(Glucopyranosyl Lipid A Adjuvant System) activates DC by binding to the TLR-4 receptor and inducing strong Th1 type CD4 responses against co-delivered recombinant proteins. Currently both platform technologies are being investigated in phase I clinical trials in cancer patients. Human carbonic anhydrase 9 (hCAIX) is a tumor-associated transmembrane antigen that is over-expressed on various cancer cell types. We mapped hCAIX specific, multi-functional CD8 and CD4 T-cell epitopes within the extracellular and transmembrane regions of the protein for the mouse haplotype H-2b by intracellular cytokine staining. Mice lethally challenged s.c. on the flank with a B16-F10 tumor cell line expressing the hCAIX protein (designated BC.12) fully controlled large tumors (>100 mm2) when therapeutically immunized (subcutaneously at the base of tail) with ZVex™ encoding hCAIX or the recombinant hCAIX protein with GLAAS™ (either s.c. or i.m.). In both models, tumor control was dose-dependent. Additionally, the presence of a strong transmembrane H-2b restricted CD8 T-cell epitope was required for tumor control and regression. hCAIX-specific CD8 T-cell responses were detectable as far out as day 67 post challenge in mice displaying full regression of tumor. These results demonstrate proof of concept for ZVex™ and GLAAS™ platform technologies in an aggressive murine melanoma model. Citation Format: David J. Campbell, Rebecca S. Reeves, Patrick A. Flynn, Scott H. Robbins, Peter Berglund, Jan H. ter Meulen. Therapeutic efficacy of the ZVex™ and GLAAS™ platforms in a B16-F10/hCAIX melanoma mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2505. doi:10.1158/1538-7445.AM2015-2505

Abstract 702: DCVex(TM): A novel integration-deficient lentivector technology that incorporates genetic and post-translational elements to target dendritic cells

With the goal of creating antigen-directed immunotherapeutics that can be safely administered directly to patients, Immune Design has developed a platform of novel integration-deficient lentiviral vectors that target and deliver antigen-encoding nucleic acids to dendritic cells (DCs) in order to promote the activation of antigen-specific effector CD8 T cells. This platform, termed DCVex(TM), utilizes a novel genetic variant of a Sindbis virus envelope glycoprotein with post-translational carbohydrate modifications in combination with Vpx, a SIVmac viral accessory protein, to achieve efficient targeting and transduction of DCs. In addition, DCVex(TM) incorporates safety features in its design that include redundant mechanisms to render DCVex(TM) integration-deficient, as well as genetic modifications that eliminate psi-gag recombination between split genome components. The characteristics that allow DCVex(TM) to specifically transduce human DCs and the advances that DCVex(TM) brings to conventional third-generation lentiviral vector design demonstrate its potential as a vaccine designed to utilize DCs for cancer immunotherapy. Citation Format: Semih U. Tareen, Brenna Kelley-Clarke, Christopher J. Nicolai, Megan M. Slough, Chintan D. Vin, Neal Van Hoeven, Scott H. Robbins, Jan H. ter Meulen, Peter Berglund. DCVex(TM): A novel integration-deficient lentivector technology that incorporates genetic and post-translational elements to target dendritic cells. [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 702. doi:10.1158/1538-7445.AM2014-702