Non-injected Tumors Research Articles

Immunostimulatory TLR7 agonist-nanoparticles together with checkpoint blockade for effective cancer immunotherapy.

Mono- or dual-checkpoint inhibitors for immunotherapy have changed the paradigm of cancer care; however, only a minority of patients responds to such treatment. Combining small molecule immuno-stimulators can improve treatment efficacy, but they are restricted by poor pharmacokinetics. In this study, TLR7 agonists conjugated onto silica nanoparticles showed extended drug localization after intratumoral injection. The nanoparticle-based TLR7 agonist increased immune stimulation by activating the TLR7 signaling pathway. When treating CT26 colon cancer, nanoparticle conjugated TLR7 agonists increased T cell infiltration into the tumors by > 4× and upregulated expression of the interferon γ gene compared to its unconjugated counterpart by ~2×. Toxicity assays established that the conjugated TLR7 agonist is a safe agent at the effective dose. When combined with checkpoint inhibitors that target programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), a 10-100× increase in immune cell migration was observed; furthermore, 100 mm3 tumors were treated and a 60% remission rate was observed including remission at contralateral non-injected tumors. The data show that nanoparticle based TLR7 agonists are safe and can potentiate the effectiveness of checkpoint inhibitors in immunotherapy resistant tumor models and promote a long-term specific memory immune function.

Antibody Opsonization of a TLR9 Agonist-Containing Virus-like Particle Enhances In Situ Immunization.

The immunologic and therapeutic effects of intratumoral (IT) delivery of a novel virus-like particle as a lymphoma immunotherapy were evaluated in preclinical studies with human cells and a murine model. CMP-001 is a virus-like particle composed of the Qβ bacteriophage capsid protein encapsulating an immunostimulatory CpG-A oligodeoxynucleotide TLR9 agonist. In vitro, CMP-001 induced cytokine production, including IFN-α from plasmacytoid dendritic cells, but only in the presence of anti-Qβ Ab. In vivo, IT CMP-001 treatment of murine A20 lymphoma enhanced survival and reduced growth of both injected and contralateral noninjected tumors in a manner dependent on both the ability of mice to generate anti-Qβ Ab and the presence of T cells. The combination of IT CMP-001 with systemic anti-PD-1 enhanced antitumor responses in both injected and noninjected tumors. IT CMP-001 alone or combined with anti-PD-1 augmented T cell infiltration in tumor-draining lymph nodes. We conclude IT CMP-001 induces a robust antitumor T cell response in an anti-Qβ Ab-dependent manner and results in systemic antitumor T cell effects that are enhanced by anti-PD-1 in a mouse model of B cell lymphoma. Early-phase clinical evaluation of CMP-001 and anti-PD-1 combination therapy in lymphoma will begin shortly, based in part on these results.

Abstract 1452: Development of ONCR-148, a miR-attenuated oncolytic HSV-1 designed to potently activate antitumor T cell response

Abstract Oncolytic viruses, through cancer cell-selective lysis and accompanying release of danger signals that promote immune activation, have demonstrated antitumor efficacy in monotherapy of metastatic melanoma and have shown promising activity in combination with checkpoint inhibitors. We present here the activity of ONCR-148, a recombinant oncolytic Herpes Simplex Virus (oHSV) designed to be a safe and efficacious therapy for the treatment of solid tumors. Neuron-specific miR attenuation of ICP4 gene inhibits viral replication in neurons while preserving its potent oncolytic activity in tumor cells. The antitumor potency of the virus is enhanced by incorporating in its genome a transgene expressing fibroblast activation protein (FAP)- and CD3- bispecific antibody that recruits and re-directs the cytolytic activity of T cells toward FAP-expressing stromal cells. FAP is a cell-surface serine protease that is almost exclusively found in fibroblasts within tumor stroma. However, systemic administration of a FAP-CD3 bispecific antibody was not tolerated due to the toxicity toward FAP-expressing bone marrow mesenchymal cells (Tran et al., 2013. J.Exp.Med, 210(6), 1125-1135). Thus, the local intra-tumoral delivery of FAP-CD3 is an attractive approach to promote tumor lysis and T cell activation against cancerous lesions. To test the in vivo activity of ONCR-148, as FAP is poorly expressed in syngeneic mouse tumor models, we developed a bilateral mouse FAP-expressing MC38 syngeneic colon carcinoma model (MC38-FAP), allowing to test local antitumor activity in oHSV-injected tumors and abscopal efficacy mediated by immune cells in the non-injected tumors. Intra-tumoral administration of ONCR-148 in the MC38-FAP model resulted in tumor growth inhibition of 60% (p<0.03) and 46% (p<0.01) on the injected (ipsilateral) and non-injected (contralateral) tumor, respectively. We then investigated additional transgenes that could potentiate the antitumor activity of ONCR-148. We combined in vivo ONCR-148 with ONCR-153, an oHSV armed with the NK and T cell activating cytokine IL-12 and the chemokines CXCL10 and CCL4, allowing for expansion and recruitment of T cells and antigen presenting cells in the tumor milieu. Combined intra-tumoral administration of ONCR-148 and ONCR-153 increased the antitumor response to 90% on the injected tumor (p<0.0001), leading to complete tumor remission in 5 out of 9 treated mice and to 80% on distant tumors (p<0.0001, with no complete responses observed).These data indicate that oHSV expressing bispecific antibodies, while being well tolerated, is efficacious in injected tumors and mediate systemic antitumor immune response. The efficacy can be potentiated by a local co-expression of immunostimulatory cytokines. Such data support the further development of oHSV-1 armed with CD3 bispecific antibodies for the therapy of patients with metastatic cancer. Citation Format: Agnieszka Denslow, Brian B. Haines, Michael S. Ball, Jacqueline Gursha, Daniel Wambua, Cecilia Kwong, Lingxin Kong, Allison Colthart, Prajna Behera, Peter Grzesik, Jennifer Lee, Terry Farkaly, Caitlin Goshert, Edward M. Kennedy, Lorena Lerner, Christophe Quéva. Development of ONCR-148, a miR-attenuated oncolytic HSV-1 designed to potently activate antitumor T cell response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1452.

Abstract CT090: Rational design of an oncolytic virus permits use of interferon beta as a pharmacodynamic marker for clinical application

Abstract Introduction. Voyager-V1 (VV1) is an oncolytic vesicular stomatitis virus engineered to express human interferon beta (IFNβ) to enhance cellular antitumor immune responses and tumor selectivity. VV1 also contains the human sodium iodide symporter (NIS) as an imaging gene. We report here the novel use of virus-encoded IFNβ as a PD marker using correlative data from three Phase I trials of VSV-IFNβ-NIS in patients with refractory cancers (n=46). Methods. 46 patients with solid tumors (n=34) and hematological malignancies (n=12) received 1 dose of VV1 either intratumorally (ITu) or intravenously (IV) at doses ranging from 3 x 106 to 5 x 1010 TCID50. Plasma IFNβ levels were collected pre-treatment, 4 hours post-infusion, Day 2 (24-hour), Day 3, 8, 15 and 29 (IT only). Samples were processed using a standard IFNβ specific ELISA kit. Results. ITu dose escalation is complete with 27 patients treated and no DLTs. IV escalation is ongoing at 5 x 1010 TCID50 with 19 patients treated to date. In the ITu study, plasma IFNβ levels at 24h were undetected at the lowest dose levels (up to 1 x 107 TCID50), and became detectable from 3 x 107 TCID50. In the IV patients, IFNβ was detectable at all dose levels (5 x 109 through 5 x 1010 TCID50) with the highest peak and longest duration in a patient with metastatic endometrial cancer coincident with shrinkage of multiple tumors. The IFNβ produced by virus-infected cells can be differentiated from the acute innate antiviral responses by magnitude of response and AUC as the majority of the inflammatory cytokines returned to baseline by 48h. Peak IFNβ levels were variable between patients, likely reflecting heterogeneity in tumor susceptibility to VV1, ranging from 1.4pg/mL to 656pg/mL across 6 patients (mean 153pg/ml) at the highest ITu dose. Plasma IFNβ 24 hours post-therapy of >20pg/mL appears to predict for RECIST 1.1-evaluated SD vs PD, p=0.048 in the ITu patients. Peak IFNβ ranged from 18 to 1700 pg/mL across 9 patients (mean=442pg/ml) at 1.7 x 1010 in the IV study. Peak IFNβ was highest in two cases of endometrial cancer (1500 and 1700 pg/ml). The patient with the highest IFNβ levels on the IV trial showed 16.7% tumor shrinkage at the first tumor evaluation. SPECT imaging, which shows location of viral replication, was positive in 50% of ITu injected tumors, also validating of VV1 infection of target cells. To date, SPECT images were negative in the IV trial despite IFNβ positivity, reflecting IFNβ as a more sensitive PD marker of viral infection. IHC staining of tumor biopsies collected pre-treatment and 1 month after VSV showed increased numbers of CD3, CD8, CD68, PDL1 or PD1 in some injected or noninjected tumors. Other immune markers and tumor gene signatures are also being evaluated. Conclusions. Plasma IFNβ has emerged as a simple and convenient biomarker of viral replication in tumors. IFNβ will be used in future studies as a PD marker to assess the impact of immune-modulating combination drugs with VV1. Citation Format: Timothy P. Cripe, Jamie Bakkum-Gamez, Jaime R. Merchan, Martha Q. Lacy, Manish R. Patel, Steven Powell, James Strauss, Lianwen Zhang, Toshie Sakuma, Memy Diaz, Nandakumar Packiriswamy, Deepak Upreti, Bethany Brunton, Dragan Jevremovic, Stephen J. Russell, Alice Bexon, Kah-Whye Peng. Rational design of an oncolytic virus permits use of interferon beta as a pharmacodynamic marker for clinical application [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT090.

Abstract 944: exoSTING: An engineered exosome therapeutic that selectively delivers STING agonist to the tumor resident antigen-presenting cells resulting in improved tumor antigen-specific adaptive immune response

Abstract Background: The Stimulator of Interferon Genes (STING) pathway is an attractive target in immuno-oncology. Selective activation of the STING pathway in antigen presenting cells (APCs) is essential for eliciting a potent and specific anti-tumor immune response. STING is ubiquitously expressed in normal cells including T cells and endothelial cells. Direct intra-tumoral administration of a free STING agonist results in activation of the STING pathway in all cells, resulting in loss of viability of immune cells, tissue damage, and systemic immune activation due to vascular leakage. Exosomes are an efficient natural messenger system that delivers macromolecules between cells. Leveraging exosome biology, we have developed a novel engineered exosome therapeutic, exoSTING, to selectively target the STING pathway in tumor resident APCs. Results: exoSTING is composed of exosomes engineered to express high levels of Protein X (PrX), a transmembrane glycoprotein naturally occurring on exosomes, and loaded with a STING agonist. Following intra-tumoral injection in checkpoint refractory B16F10 tumors, exoSTING selectively activates STING in APCs, leading to anti-tumor immunity with greater than 100-fold improvement in potency compared to free STING agonist. The activity of exoSTING is substantially diminished in the PrX knock out exosomes, highlighting the importance of this surface glycoprotein for preferential activation of APCs. In contrast to the free STING agonist, exoSTING is retained within the injected tumor, minimizing systemic exposure. Furthermore, exoSTING administration preserves the viability of T cells and APCs, reduces collateral tissue damage, and does not induce systemic cytokine production, resulting in a broader therapeutic window in contrast to free STING agonist. exoSTING produced an increased systemic tumor-specific T cell response as demonstrated by elimination of non-injected abscopal tumors. The specificity of exoSTING activity was demonstrated using a STING knockout mouse (Tmem173gt/J). T cells and myeloid cells play a critical role in exoSTING mediated anti-tumor immunity, in contrast to NK cells which are not required. exoSTING treatment results in significant induction of PD-L1 expression (P<0.005). In combination with PD1 checkpoint blockade, exoSTING shows enhanced efficacy over high-dose free STING agonist on abscopal tumors. Conclusion: exoSTING is an engineered exosome therapeutic that leverages exosome biology and specifically targets the STING pathway in APCs, resulting in greater potency with preserved viability of T cells and APCs, greater systemic tumor antigen specific immune response, reduced systemic cytokine production, and enhanced efficacy. Citation Format: Su Chul Jang, Raymond J. Moniz, Chang Ling Sia, Rane A. Harrison, Damian Houde, Nikki Ross, Ke Xu, Nuruddeen Lewis, Raymond Bourdeau, Christine McCoy, Tong Zi, Agata Villiger-Oberbek, Scott Estes, Jorge Sanchez-Salazar, Kevin Dooley, William K. Dahlberg, Sriram Sathyanarayanan, Kyriakos D. Economides. exoSTING: An engineered exosome therapeutic that selectively delivers STING agonist to the tumor resident antigen-presenting cells resulting in improved tumor antigen-specific adaptive immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 944.

Phase Ib study of MIW815 (ADU-S100) in combination with spartalizumab (PDR001) in patients (pts) with advanced/metastatic solid tumors or lymphomas.

2507 Background: MIW815 (ADU-S100) is a novel synthetic cyclic dinucleotide that activates the STimulator of INterferon Genes (STING) pathway impacting tumor cells, tumor microenvironment, vasculature, tumor-associated fibroblasts, and priming APC and CD8+ T cells. Spartalizumab is a humanized IgG4 mAb that blocks the binding of PD-1 to PD-L1/2. Preclinical data support synergistic antitumor effects when MIW815 (ADU-S100) is combined with checkpoint inhibitors. Methods: In this Phase Ib dose escalation study, pts with advanced/metastatic solid tumors or lymphoma received MIW815 (ADU-S100) (intratumoral injections [50–800 µg] either weekly [3 weeks on/1 week off] or Q4W) and spartalizumab (400 mg IV Q4W). Injected and non-injected tumor biopsies were obtained at baseline and on treatment. Primary objectives are to determine safety and identify a dose/schedule for future studies. Preliminary activity, pharmacokinetics (PK), and pharmacodynamics (PD) are also being explored. Results: As of Jan 11, 2019, 66 pts (median age: 61 y) with various solid tumors or lymphomas have been treated. Treatment was discontinued in 49 pts (74%) due to disease progression (n = 28), pt/physician decision (n = 18), AE (n = 2), or death (n = 1). No DLTs were reported during the first cycle at any dose level. Most common (≥5 pts) treatment-related AEs (TRAEs) were injection site pain (12%), pyrexia (11%), and diarrhea (9%). Grade 3/4 TRAEs (in ≥2 pts) were increased AST and ALT (3% each). Serious TRAEs were pyrexia (3%), increased amylase, increased lipase, diarrhea, fatigue, hyperthyroidism, partial seizures, dyspnea, and pneumonitis (all 2%). Partial responses in pts with PD-1–naive TNBC and PD-1–relapsed/refractory melanoma have been observed. MIW815 (ADU-S100) plasma exposure generally increased in a dose-dependent manner with a rapid terminal half-life. Response data, PK and PD analyses will be presented. Conclusions: Thus far, MIW815 (ADU-S100) + spartalizumab has demonstrated antitumor activity in PD-1–naive TNBC and PD-1–relapsed/refractory melanoma. The combination is well tolerated, with no DLTs reported to date. The MTD has not been reached and dose escalation is ongoing. Clinical trial information: NCT03172936.

Immunotherapeutic effects of intratumoral nanoplexed poly I:C

Poly I:C is a powerful immune adjuvant as a result of its agonist activities on TLR-3, MDA5 and RIG-I. BO-112 is a nanoplexed formulation of Poly I:C complexed with polyethylenimine that causes tumor cell apoptosis showing immunogenic cell death features and which upon intratumoral release results in more prominent tumor infiltration by T lymphocytes. Intratumoral treatment with BO-112 of subcutaneous tumors derived from MC38, 4 T1 and B16-F10 leads to remarkable local disease control dependent on type-1 interferon and gamma-interferon. Some degree of control of non-injected tumor lesions following BO-112 intratumoral treatment was found in mice bearing bilateral B16-OVA melanomas, an activity which was enhanced with co-treatment with systemic anti-CD137 and anti-PD-L1 mAbs. More abundant CD8+ T lymphocytes were found in B16-OVA tumor-draining lymph nodes and in the tumor microenvironment following intratumoral BO-112 treatment, with enhanced numbers of tumor antigen-specific cytotoxic T lymphocytes. Genome-wide transcriptome analyses of injected tumor lesions were consistent with a marked upregulation of the type-I interferon pathway. Inspired by these data, intratumorally delivered BO-112 is being tested in cancer patients (NCT02828098).

Oncolytic virus immunotherapy induces immunogenic cell death and overcomes STING deficiency in melanoma

ABSTRACTSuccessful immunotherapy for melanoma depends on the recruitment of effector CD8+ T cells to the tumor microenvironment. Factors contributing to T cell regulation in melanoma have recently been recognized, including the stimulator of interferon genes (STING). Agents that can activate STING or enhance T cell infiltration into established tumors have become an important focus for further clinical development. Talimogene laherparepvec (T-VEC) is an oncolytic herpes simplex virus, type 1 (HSV-1) encoding granulocyte-macrophage colony stimulating factor (GM-CSF) and is approved for the treatment of melanoma and has shown therapeutic activity in murine tumors known to express high levels of STING. The mechanism of action for T-VEC has not been fully elucidated but is thought to include induction of immunogenic cell death (ICD) and activation of host anti-tumor immunity. Thus, we sought to investigate how T-VEC mediates anti-tumor activity in a melanoma model. To determine if T-VEC induced ICD we established the relative sensitivity of a panel of melanoma cell lines to T-VEC oncolysis. Following T-VEC infection in vitro, melanoma cell lines released of HMGB1, ATP, and translocated ecto-calreticulin. To identify potential mediators of this effect, we found that melanoma cell sensitivity to T-VEC was inversely related to STING expression. CRISPR/Cas9-STING knockout was also associated with increased T-VEC cell killing. In the D4M3A melanoma, which has low expression of STING and is resistant to PD-1 blockade therapy, T-VEC was able to induce therapeutic responses in both injected and non-injected tumors and demonstrated recruitment of viral- and tumor-antigen specific CD8+ T cells, and induction of a pro-inflammatory gene signature at both injected and non-injected tumors. These data suggest that T-VEC induces ICD in-vitro and promotes tumor immunity and can induce therapeutic responses in anti-PD-1-refractory, low STING expressing melanoma.

Radioguided occult lesion localization in patients with recurrent thyroid cancer.

Well planned re-surgery is critical for recurrent/persistent thyroid cancer (TC). We aimed to investigate the clinical outcome of radioguided-occult lesion localization (ROLL) guided surgery in patients with recurrent/persistent TC. This study includes 29 patients [F/M: 22/7, median age 43 ± 12years (18-58)] with a diagnosis of TC (22 papillary, 2 follicular and 5 medullary). Before surgery, all patients underwent ultrasonography (USG) guided mapping and intra-lesional radioactivity injection. Surgery was performed based on the excision of radioactivity injected lesions by a gamma probe and non-injected tumor foci via USG-neck map. Researchers determined surgical success by post-operative tumor markers and neck-USG. Among 29 patients, 60 metastatic lesions were identified by USG [median size 10 ± 6.3mm (range 5-30)]. Neck-USG performed after surgery provided no evidence of disease (NED) in %97 (28/29) of TC patients. In the follow-up, stimulated thyroglobulin (Tg) levels were less than 1ng/ml in 79%(19/24) of DTC patients and suppressed Tg < 0.2ng/ml was noted in 92% (22/24).In patients with DTC with an incomplete structural response, we dramatically changed the American Thyroid Association (ATA) response category and achieved an excellent response in 92% (22/24) of patients. Among patients with MTC, 5/5 patients had normal USG and calcitonin levels were reduced by 60-80% in 4/5 and > 80% in1/5 patients. In this study we have shown that, ROLL-guided surgery yielded NED rate of %97 (28/29) and increased excellent response rates according to ATA guidelines. Further studies with larger patient groups and longer follow-up should be performed to confirm the efficacy of this surgery.

Abstract PR09: Intratumoral delivery of engineered modified vaccinia virus Ankara expressing Flt3L and OX40L for "in situ" therapeutic cancer vaccination

Abstract Modified vaccinia virus Ankara (MVA) is a highly attenuated, effective, and safe vaccinia strain that is an important vaccine vector for infectious diseases and cancers. We have previously shown that intratumoral (IT) injection of inactivated MVA elicits stronger antitumor immunity compared with live MVA in murine B16-F10 melanoma and MC38 colon cancer models, indicating that viral immune inhibitory factors might be involved in attenuating antitumor effects. Here we provide evidence that vaccinia virulence factor C7 interacts with transcription factors IRF3 and STAT2, which attenuates both type I IFN production and IFN receptor signaling. Infection of conventional dendritic cells (cDCs) with MVAΔC7L in which C7L gene is deleted results in higher levels of IFNB gene induction and IRF3 phosphorylation compared with MVA. IT MVAΔC7L also induced stronger antitumor responses compared with MVA, which correlates with higher levels of tumor infiltrating activated CD4+ and CD8+ T-cells in both injected and noninjected tumors in a bilateral B16-F10 tumor implantation model. We engineered a recombinant MVA in which the C7L gene was replaced with human Flt3L (hFlt3L) and the J2R gene (a.k.a. TK gene) was replaced with murine OX40L (mOX40L) or human OX40L (hOX40L). Using B16-F10 and MC38 tumor models, we show that IT MVAΔC7L-hFlt3L-ΔJ2R-mOX40L is more effective than MVAΔC7L-hFlt3L, or MVAΔC7L, or heat-inactivated MVAΔC7L in eradicating injected tumors, delaying the growth of the noninjected tumors, and prolonging mice survival. ELISPOT analysis demonstrate that IT MVAΔC7L-hFlt3L-ΔJ2R-mOX40L generates the most tumor-specific CD8+ and CD4+ T-cells compared with the other viruses mentioned above. The combination of IT MVAΔC7L-hFlt3L-ΔJ2R-mOX40L with systemic delivery of anti-CTLA-4 or anti-PD-L1 antibodies results in more efficient eradication of injected tumors, higher survival rate compared with IT virus alone in both bilateral tumor implantation and unilateral large established tumor models. MVAΔC7L-hFlt3L-ΔJ2R-hOX40L has been generated and will be investigated in clinical trials for patients with metastatic cancers in the near future. Taken together, we provide proof-of-concept results for the development of immune activating recombinant MVA to alter tumor immunosuppressive microenvironment through the induction of type I IFN via the cytosolic DNA-sensing pathway, activation of CD103+ dendritic cells, as well as direct activation of both CD4 and CD8 T-cells. This approach enhances tumor antigen presentation and T-cell activation through "in situ" therapeutic vaccination effects, either used as monotherapy or in combination with anti-CTLA-4 or anti-PD-L1 antibody therapy. Citation Format: Liang Deng, Ning Yang, Yi Wang, Wei Yan, Jiahu Wang, John Choi, Stewart Shuman, Taha D. Merghoub, Jedd D. Wolchok. Intratumoral delivery of engineered modified vaccinia virus Ankara expressing Flt3L and OX40L for "in situ" therapeutic cancer vaccination [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr PR09.

Intratumoral Injection of CpG-ODN Plus Systemic Ibrutinib Induces an Anti-Tumor Immune Response Affecting T Cell Subsets in the Microenvironment of Both Injected and Non-Injected Tumor Sites in Patients with Low-Grade Lymphoma

BACKGROUND: Low-grade B cell lymphoma is often characterized by an infiltration of immune effector cells including T, NK and dendritic cells. But, despite the presence of effector cells within the tumor, these cells fail to control tumor growth.In a preclinical mouse model, we showed that Ibrutinib, an inhibitor of Bruton's tyrosine kinase (BTK), but also ITK (IL-2-inducible T cell kinase), synergized with intratumoral CpG to facilitate complete regression of tumors at the treated site as well as a distal, non-treated site, curing all the mice. This was accompanied by a potent anti-tumor memory T cell response by both CD4 and CD8 T cells that rejected the tumor on re-challenge. (Sagiv-Barfi I, et al. Blood. 2015 March:125(13):2079-2086)STUDY: In an ongoing clinical trial (NCT02927964), patients with previously treated low-grade lymphoma receive low dose (2Gyx2) radiotherapy to a single tumor site followed by 5 weekly intratumoral injections of 3 mg CpG-ODN (SD-101, Dynavax Technologies) into the same site. 1 day after the second injection, patients begin taking a daily 560 mg dose of Ibrutinib. A fine needle aspirate (FNA) of the injected site and a non-injected site outside the radiation field is performed prior to radiotherapy, one week after the first injection, and at week 6, 1 week after the final injection of CpG. FNA samples are stained for flow cytometry with panels of antibodies to delineate all major cell populations and their subsets. Cellular activation as well as T cell exhaustion, inhibition and function are also characterized. When feasible, a biopsy is performed prior to treatment providing tumor cells to be used in an immune response assay to evaluate induced anti-tumor responses by circulating peripheral blood T cells obtained throughout the study.RESULTS: To date, 12 patients have been entered onto the study. Of these patients, 4 had excisional biopsies and subsequent immune response assays performed. All 4 patients exhibited CD8 anti-tumor responses as determined by an increase in the activation marker CD137 as well as the functional marker granzyme B above pretreatment CD8 T cells. At week 12, 7 weeks after the last CpG injection, the average increase above pretreatment baseline for CD137 and granzyme B was 6.1% and 9.3%, respectively. In 3 of these patients, we observed an increase in CD8 T cells expressing CD137 and granzyme B from the FNA of the non-injected site. The 4th patient did not have adequate number of cells for staining. Two patients exhibited CD4 immune responses as characterized by an up-regulation of CD137 and CD278.FNA of 7 patients produced enough cells to analyze the tumor microenvironment from both the injected and the non-injected sites over all 3 time points; 1 patient was evaluable through week 2 for both sites. In 7 out of these 8 patients, CD3 T cells increased at the injected and non-injected sites by week 6. The proportion of CD4 and CD8 T cells did not stay constant, however, as reflected by the changes in CD4:CD8 ratios. This suggests that the increase in T cells was not purely the result of a loss of tumor B cells in the samples. Notably, we observed a significant increase in the effector CD4 T cells in all patients at the injected site by week 2 (14.1% ± 6.5%, p = 0.005) and 5 of 8 patients in the non-injected site by week 6 (12.4% ± 10.4%). Moreover, the proportion of T follicular helper cells significantly decreased in all patients at the treated site by week 2 (17.7% ± 9.7% of T cells, p = 0.0012) and in 5 of 8 patients at the non-treated site by week 6 (8.1% ± 5.5% of T cells). Tregs were more variable, decreasing in 5 of 8 patients in the treated and 3 of 8 patients in the non-treated site by week 6.CONCLUSION: CpG is known to activate antigen-presenting cells such as dendritic cells and macrophages. Ibrutinib is a small molecule that has been shown to have direct anti-tumor effects in B cell lymphoma and may skew an immune response towards that of a TH1 type. Here we show that together, they can effect changes in the tumor microenvironment in both treated and in untreated sites of disease.This clinical trial is ongoing and open to accrual. DisclosuresKhodadoust:Innate Pharma: Research Funding.

Abscopal Effect in Non-injected Tumors Achieved with Cytokine-Armed Oncolytic Adenovirus

Cancer treatment with local administration of armed oncolytic viruses could potentially induce systemic antitumor effects, or the abscopal effect, as they self-amplify in tumors, induce danger signaling, and promote tumor-associated antigen presentation. In this study, oncolytic adenovirus coding for human tumor necrosis factor alpha (TNF-α) and interleukin-2 (IL-2) Ad5/3-E2F-d24-hTNF-α-IRES-hIL-2 (also known as [a.k.a.] TILT-123) provoked antitumor efficacy in tumors that were injected with Ad5/3-E2F-d24-hTNF-α-IRES-hIL-2 and those that were left non-injected in the same animal. Importantly, the virus was able to travel to distant tumors. To dissect the effects of oncolysis and cytokines, we studied replication-incompetent viruses in mice. Systemic antitumor effects were similar in both models, highlighting the importance of the arming device. The cytokines induced positive changes in immune cell infiltrates and induced the expression of several immune-reaction-related genes in tumors. In addition, Ad5/3-E2F-d24-hTNF-α-IRES-hIL-2 was able to increase homing of adoptively transferred tumor-infiltrating lymphocytes into both injected and non-injected tumors, possibly mediated through chemokine expression. In summary, local treatment with Ad5/3-E2F-d24-hTNF-α-IRES-hIL-2 resulted in systemic antitumor efficacy by inducing immune cell infiltration and trafficking into both treated and untreated tumors. Moreover, the oncolytic adenovirus platform had superior systemic effects over replication-deficient vector through spreading into distant tumors.

1246PD - Talimogene laherparepvec (T-VEC) treatment increases intratumoral effector T-cell and natural killer (NK) cell density in noninjected tumors in patients (pts) with stage IIIB–IVM1c melanoma: Evidence for systemic effects in a phase II, single-arm study

1246PD - Talimogene laherparepvec (T-VEC) treatment increases intratumoral effector T-cell and natural killer (NK) cell density in noninjected tumors in patients (pts) with stage IIIB–IVM1c melanoma: Evidence for systemic effects in a phase II, single-arm study

Abstract LB-306: Oncolytic vaccinia virus expressing immune checkpoint blockade antibody as cancer immunotherapeutics

Abstract Oncolytic viruses can be engineered to become multifunctional cancer immunotherapeutic agents, used either as monotherapy or in combination with immune checkpoint blockade for antitumor effects. Intratumoral (IT) delivery of oncolytic viruses expressing immunomodulatory agents can alter the tumor immunosuppressive microenvironment and facilitate the proliferation and activation of antitumor effector and memory T cells. Although anti-cytotoxic T lymphocyte protein 4 (CTLA-4) antibody has been approved for the treatment of advanced melanoma, its toxicity profile and modest efficacy as a single agent have limited its use in the clinic. Poxviruses are large cytoplasmic DNA viruses and vaccinia virus is a prototypic poxvirus that has been investigated intensively as an oncolytic virus. Its 200-kb genome size allows large insertions of multiple foreign genes. Vaccinia virus has been used in humans extensively in the past during smallpox vaccination. In this study, we engineered a vaccinia (Western Reserve)-based oncolytic virus that expresses anti-muCTLA-4 antibody and human FMS-like tyrosine kinase 3 ligand (Flt3L) for the treatment of murine implantable melanoma via intratumoral injection. We used a mutant vaccinia virus E3LΔ83N as the parental virus. This virus has a deletion of the Z-DNA-binding domain of E3, a key virulence factor, which results in 1000-fold attenuation of the virus. Through homologous recombination at the thymidine kinase (TK) locus of vaccinia E3LΔ83N virus, we successfully generated an attenuated recombinant virus (E3LΔ83N-TK--hFlt3L-anti-muCTLA-4) with a deletion of TK and an insertion of a cassette that allows the expression of both hFlt3L and anti-muCTLA-4 antibody under the vaccinia synthetic early and late promoter. This virus replicates in murine and human tumor cell lines and expresses desired anti-muCTLA-4 antibody and hFlt3L in murine and human melanoma cell lines. IT delivery of this recombinant vaccinia virus is more efficacious compared with E3LΔ83N-TK- (used either alone or in combination of systemic delivery of anti-CTLA-4 antibody), in eradicating or delaying the growth of both injected tumors and non-injected distant tumors, as well as in prolonging the survival of mice in a murine bilateral B16-F10 melanoma tumor implantation model. Furthermore, immunological analyses of tumor-infiltrating lymphocytes, as well as antitumor T cells in spleens, showed highest numbers of activated Granzyme B+ CD4+ and CD8+ T cells in the non-injected distant tumors and highest numbers of antitumor CD8+ T cells in the spleens of mice treated with IT delivery of E3LΔ83N-TK--hFlt3L-anti-muCTLA-4 compared with those treated with E3LΔ83N-TK--hFlt3L or E3LΔ83N-TK-. No toxicities related to IT E3LΔ83N-TK--hFlt3L-anti-muCTLA-4 have been observed. Taken together, our results demonstrate that IT delivery of oncolytic vaccinia virus expressing anti-CTLA-4 and hFlt3L is a safe and effective strategy to enhance antitumor immunity. Citation Format: Weiyi Wang, Peihong Dai, Ning Yang, Stewart Shuman, Wei Yan, Taha Merghoub, Jedd D. Wolchok, Liang Deng. Oncolytic vaccinia virus expressing immune checkpoint blockade antibody as cancer immunotherapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-306.

Abstract 4698: ONCR-1, a novel herpes simplex virus expressing MMP9 and ULBP3 transgenes, evokes potent oncolysis and development of antitumor immune responses

Abstract Glioblastoma multiforme (GBM) is a highly aggressive brain tumor associated with poor prognosis and resistance to current therapies. ONCR-1 is a novel oncolytic herpes simplex virus type-1 vector in development for the treatment of GBM. ONCR-1 utilizes a unique conditional-lethal strategy in which miR124 binding sites are inserted into the ICP4 locus to prevent viral replication in neuronal cells while preserving one copy of the γ34.5 gene and enabling potent cytotoxicity in tumor cells. ONCR-1 is armed with a matrix metalloproteinase 9 (MMP9) transgene to facilitate viral spread in the tumor extracellular matrix. Further, ONCR-1 expresses a UL16 binding protein 3 (ULBP3) transgene to activate NK and CD8+ T cells via the NKG2D receptor and promote antitumor immune responses. In human and murine tumor cells in vitro, ONCR-1 evoked potent cytotoxicity. ONCR-1 increased proteolytic MMP in cell supernatants and ULBP3 cell surface expression with no effect on related NKG2D ligands. In mice bearing subcutaneous or orthotopic human U251 GBM tumors, ONCR-1 administered intratumorally (3x105-3x106 PFU QDx1) inhibited tumor growth and prolonged survival. Ex vivo analysis of immune cells in subcutaneous tumors showed significant increases in virally evoked NKG2D+ NK cells with ONCR-1 treatment. In mice bearing dual flank subcutaneous syngeneic A20 tumors, ONCR-1 significantly inhibited tumor growth in injected tumors (1x105-3x105 PFU, Q2Dx3) and evoked potent systemic antitumor immune responses as evidenced by significant inhibition of distal noninjected left flank tumors. IFNγ production from isolated splenocytes cultured in the absence and presence of A20 tumor cells was significantly augmented in ONCR-1-treated animals, providing further evidence of an enhanced antitumor immune response. Moreover, in mice bearing subcutaneous A20 tumors in which ONCR-1 evoked complete tumor regression, complete protection from rechallenge with A20 tumor cells was observed. Depletion of NK cells or CD8+ T cells in mice resulted in inhibition of ONCR-1-mediated antitumor effects in both injected and noninjected murine A20 tumors, supporting an immune mechanism of action. Our preclinical studies demonstrate that ONCR-1 evokes tumor cell killing by direct oncolysis and by enhancing antitumor immune responses. ONCR-1 represents a novel clinical candidate for the treatment of GBM. Citation Format: Alexandra Hicks, Paola Grandi, Michael Paglia, Jingzang Miu, Cecilia Kwong, Jacqueline Gursha, Michael Ball, Weiguo Yao, Daniel Wambua, Terry Farkaly, Kyle Grant, Laura Viggiano Salta, Lorenz Ponce, Joseph Glorioso, Christophe Queva, Mitchell Finer. ONCR-1, a novel herpes simplex virus expressing MMP9 and ULBP3 transgenes, evokes potent oncolysis and development of antitumor immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4698.

Abstract CT144: Intratumoral toll-like receptor 9 (TLR9) agonist, CMP-001, in combination with pembrolizumab can reverse resistance to PD-1 inhibition in a phase Ib trial in subjects with advanced melanoma

Abstract Background: CMP-001 comprises a CpG-A oligodeoxynucleotide packaged within a virus-like particle. It is designed to activate tumor-associated plasmacytoid dendritic cells via TLR9 inducing an interferon-rich tumor microenvironment and anti-tumor CD8+ T cell responses. Materials and Methods: CMP-001-001 is an ongoing phase Ib trial evaluating intratumoral (IT) CMP-001 in combination with pembrolizumab (administered per label) in subjects with advanced melanoma resistant (either did not respond or progressed) on prior anti-PD-1 monotherapy or in combination. During dose escalation, subjects were enrolled to cohorts of ≥ 3 subjects at CMP-001 doses of 1, 3, 5, 7.5, and 10 mg in two dosing schedules (weekly for 7w, followed by q3w; or weekly for 2w, followed by q3w). CMP-001 was administered IT into an accessible lesion(s), and response assessed in all target lesions (injected and non-injected) by RECIST v1.1. Study therapy was continued until progression, toxicity, investigator decision or withdrawal of consent. Baseline and on-therapy serum was collected for cytokine analysis. Immunohistochemical and RNA-Seq analysis was performed on available pre- and post-treatment tumor biopsies. Results: As of December 31, 2017, 68 subjects have been treated (44 in Escalation and 24 in Expansion). Safety data from 63 subjects demonstrated a manageable acute toxicity profile consisting predominately of fever, N/V, headache, hypotension and rigors. Grade 3/4 related AEs reported in ≥1 subject; hypotension (n=7), anemia (n=2), chills (n=2), hypertension (n=2) and fever (n=2). The Objective Response Rates (ORR) across all dose cohorts on weekly (n=40) and q3week schedules (n=13) were 22.5% (9/40; 95 % CI 11-39%) and 7.7%% (1/13; 95% CI 0-36%) respectively. For subjects dosed weekly at 3 and 5 mg, the ORR was 33.3% (6/18 95% CI 13-59%). Of the 10 responders, 1 progressed (w36), 2 withdrew consent (w13, w25), 7 remain on study with 2 subjects maintaining their response though w72. Regression of non-injected tumors occurred in cutaneous, nodal, hepatic, and splenic metastases. CMP-001 induced TLR9 activation with a median 5.9 fold increase in serum CXCL10 (range of 0.9 - 276.3; mean fold increase of 21.8 with SD=48.8; n=39). Immunohistochemical and RNA-Seq analysis of tumor biopsies revealed increases in tumor-infiltrating CD8 T cells (&amp;gt;5 fold), PD-L1 expression (&amp;gt;3 fold increase in H score), and transcriptional signature of inflammation in 2/4 subjects with analyzable pre-and post-treatment samples. Conclusions: CMP-001 in combination with pembrolizumab resulted in objective, durable tumor responses with tolerable toxicities in subjects with advanced melanoma resistant to prior anti-PD-1 therapy. CMP-001 dosing at 5 mg/weekly has been selected for further evaluation in the ongoing dose expansion phase of this study. Citation Format: Mohammed Milhem, Rene Gonzales, Theresa Medina, John M. Kirkwood, Elizabeth Buchbinder, Inderjit Mehmi, Jiaxin Niu, Montaser Shaheen, Ryan Weight, Kim Margolin, Jason Luke, Aaron Morris, David Mauro, Arthur M. Krieg, Antoni Ribas. Intratumoral toll-like receptor 9 (TLR9) agonist, CMP-001, in combination with pembrolizumab can reverse resistance to PD-1 inhibition in a phase Ib trial in subjects with advanced melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT144.

Abstract 631: Intratumoral activation of STING with a synthetic cyclic dinucleotide elicits antitumor CD8 T-cell immunity that effectively combines with checkpoint inhibitors

Abstract Activation of the STING pathway by intratumoral (IT) injection of synthetic cyclic dinucleotides (CDNs) induces stable tumor regression in preclinical models, yet the underlying immune correlates are not fully understood. ADU-S100, a CDN under clinical evaluation, was administered IT with an optimized dosing regimen to explore the immune requirements for antitumor efficacy in mouse syngeneic tumor models. We show that CD8+ T cells are necessary and sufficient for durable antitumor immunity elicited by ADU-S100 and that activation of STING in hematopoietic cells mediates CD8+ T cell induction. Both type I IFN and TNFα, which are induced by STING pathway activation, influence the antitumor immune response. The combination of ADU-S100 and anti-PD1 treatment enhances CD8+ T cell-dependent, noninjected tumor control that correlates with an enhanced effector profile of CD8+ T cells in the tumor. Combination of ADU-S100 with checkpoint inhibition also enhances durable immunity in a poorly immunogenic tumor model. Together, these results elucidate the immune correlates to STING-mediated antitumor efficacy and highlight the potential of combining STING agonists with checkpoint inhibition in the clinic. Citation Format: Anthony L. Desbien, Kelsey Sivick Gauthier, Leticia Corrales, Gabrielle Reiner, Laura Hix Glickman, George Katibah, Thomas E. Hudson, Uyen Vu, Natalie H. Surh, Brian Francica, Weiwen Deng, David B. Kanne, Justin J. Leong, Chudi Ndubaku, Ken Metchette, Jeffery M. McKenna, Steven L. Bender, Meredith L. Leong, Thomas W. Dubensky Jr., Andrea van Elsas, Sarah M. McWhirter. Intratumoral activation of STING with a synthetic cyclic dinucleotide elicits antitumor CD8 T-cell immunity that effectively combines with checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 631.

Phase I/II dose escalation and expansion cohort safety and efficacy study of image guided intratumoral CD40 agonistic monoclonal antibody APX005M in combination with systemic pembrolizumab for treatment naive metastatic melanoma.

TPS3133 Background: Checkpoint blockade has become a major modality in the treatment of metastatic melanoma (MM). However, durable remission rates remain low. CD40 activation on antigen presenting cells (APCs) initiates their ability to prime and activate CD8+ T-cells through upregulation of co-stimulatory molecules (ie. CD80, CD86) as well as expression of effector cytokines. Furthermore, CD40 activation cause macrophages to develop a tumoricidal phenotype and increase MHC I expression on tumor cells. Direct IT immune modulation utilizes the tumor as a “vaccine site” to generate a tumor specific immune response. We hypothesize that IT injection of a CD40 Agonist (A), will “immunize” patients (Pts) against melanoma neoantigens through “licensing” of APCs for tumor specific T-cell priming and activation. In mouse models, we have shown that IT administration of the recombinant adenovirus encoding CD40L induces CD8+ T-cell-mediated activity against B16 melanoma and, importantly, also augmented the activity of anti-PD-1. Methods: This phase I/II trial (NCT02706353) evaluates the safety, efficacy, and immunological impact of IT administration of APX005M (CD40 A mAb) in combination with pembrolizumab in pts with MM. The phase I escalation is an accelerated 3+3 design. Pts will receive IT APX005M every 3 weeks for a total of 4 doses. Image guidance will allow for injection of visceral, nodal, and soft tissue metastases. The single-arm phase 2 expansion will evaluate the overall response rate (ORR) 12 weeks after treatment initiation. Key inclusion criteria: confirmed, measurable, metastatic cutaneous or mucosal melanoma; and at least 2 injectable lesions. Key exclusion criteria: prior immunotherapy, uveal melanoma, or active autoimmune disease. 26 pts will have 75% power to detect an improvement from a null ORR of 33% to 55%, using a one group chi-square test and assuming a one-sided α–level of 5%. Immune analysis will be performed on pre and on-treatment tumor/liquid biopsies both in injected and non-injected tumors. Dose level 3 of escalation phase has completed with 5 pts enrolled. Clinical trial information: NCT02706353.

020 The combination of intratumoral delivery of inactivated modified vaccinia virus Ankara with systemic delivery of immune checkpoint blockade enhances antitumor immunity

Preclinical and clinical studies have shown that viral-based immunotherapy has the potential to overcome resistance to immune checkpoint blockade (ICB) and to fill the unmet needs of many cancer patients. Poxviruses, such as modified vaccinia virus Ankara (MVA), have the potential as cancer immunotherapeutic agents. We recently showed that intratumoral (IT) delivery of inactivated modified vaccinia virus Ankara (iMVA) induces antitumor systemic immunity via the STING-mediated cytosolic DNA-sensing pathway and Batf3–dependent CD103+/CD8+ dendritic cells (DCs). The combination of IT iMVA and systemic delivery of ICB is highly effective in treating large established tumors and distant metastasis (Dai et al., Science Immunology, 2017). In this study, we investigated the immunological mechanisms underlying the superiority of combination therapy over monotherapy with either IT Heat-iMVA or with systemic ICB alone. Using a bilateral tumor implantation model, in which only the larger tumors were injected with IT iMVA, we found that the combination therapy with systemic ICB induced higher numbers of activated CD8+ and CD4+ T cells and higher levels of type I IFN and proinflammatory cytokines and chemkines in both injected and non-injected tumors, and more antitumor-specific memory CD8+ T cells in the spleens of mice compared with IT iMVA alone. In addition, concomitant IT iMVA and IT ICB at one ten of the dose used for systemic delivery elicited superior antitumor effects compared with IT iMVA alone. In conclusion, our results demonstrate that IT iMVA generates systemic antitumor effector and memory T cell responses via in situ vaccination, which can be augmented in the presence of ICB therapy.

Cancer terminator viruses (CTV): A better solution for viral-based therapy of cancer.

In principle, viral gene therapy holds significant potential for the therapy of solid cancers. However, this promise has not been fully realized and systemic administration of viruses has not proven as successful as envisioned in the clinical arena. Our research is focused on developing the next generation of efficacious viruses to specifically treat both primary cancers and a major cause of cancer lethality, metastatic tumors (that have spread from a primary site of origin to other areas in the body and are responsible for an estimated 90% of cancer deaths). We have generated a chimeric tropism-modified type 5 and 3 adenovirus that selectively replicates in cancer cells and simultaneously produces a secreted anti-cancer toxic cytokine, melanoma differentiation associated gene-7/Interleukin-24 (mda-7/IL-24), referred to as a Cancer Terminator Virus (CTV) (Ad.5/3-CTV). In preclinical animal models, injection into a primary tumor causes selective cell death and therapeutic activity is also observed in non-injected distant tumors, that is, "bystander anti-tumor activity." To enhance the impact and therapeutic utility of the CTV, we have pioneered an elegant approach in which viruses are encapsulated in microbubbles allowing "stealth delivery" to tumor cells that when treated with focused ultrasound causes viral release killing tumor cells through viral replication, and producing and secreting MDA-7/IL-24, which stimulates the immune system to attack distant cancers, inhibits tumor angiogenesis and directly promotes apoptosis in distant cancer cells. This strategy is called UTMD (ultrasound-targeted microbubble-destruction). This novel CTV and UTMD approach hold significant promise for the effective therapy of primary and disseminated tumors.